Responsive Nanomaterial Delivery Systems for Pancreatic Cancer Management

Pancreatic cancer is characterized by a high mortality rate and unfavorable prognosis. This is primarily attributed to poor accumulation of therapeutic agents at the target site due to the presence of a highly complex tumor microenvironment surrounding the pancreatic cancer tissue. However, a promising avenue for targeted drug delivery has emerged in the form of stimuli‐responsive materials. These advanced nanocarriers, encompassing both external and internal stimuli‐responsive nanoparticles, can be formulated to control the release of therapeutic agents precisely in response to specific activation. By harnessing external stimuli such as light, ultrasound, or magnetic fields, as well as intrinsic biological triggers including pH, redox potential, hypoxia, and temperature, these nanomaterials exhibit ‘intelligent’ and selective responses within complex biological environments. These responsive nanoparticles have been shown to address challenges associated with poor vascularity and thick desmoplastic stromal layers, which are hallmarks of pancreatic cancer, by promoting enhanced drug accumulation and release at the target site relative to conventional therapy. This work explores the design strategies for advanced stimuli‐responsive nanomaterials, integrating both internal and external stimuli, with the potential to enhance drug delivery efficacy in pancreatic cancer. It addresses the challenges and prospects in their development and offers insights for future clinical applications.


Introduction
According to the World Health Organization (WHO) GLOBOCAN database, pancreatic cancer is considered the seventh leading cause of cancer deaths worldwide, with ≈466 000 recorded deaths in 2020. [1,2]At present, a high mortality rate is associated with this disease, with patients exhibiting a five-year survival rate of only 9.3%. [3]Most patients are already at an advanced disease stage at the time of diagnosis [4] and the resultant tumor invasion and metastases can lead to a further decrease in the five-year survival rate to 3%, resulting in pancreatic cancer exhibiting the poorest prognosis of all solid tumors. [5]The most common type of pancreatic cancer is pancreatic ductal adenocarcinoma (PDAC), accounting for 85-90% of all pancreatic cancer cases. [6]Currently, a combination of surgical resection, chemotherapy, and radiotherapy is used to treat PDAC. [7][5] As such, chemotherapy Table 1.3] Treatment Name Drug Dose and route Days of administration Length of cycle Gemcitabine monotherapy Gemcitabine 1000 mg m −2 IV Every week for 7 weeks followed by 1 week rest.
Every week for 3 weeks followed by 1 week rest ease management with several agents being used, including 5-fluorouracil (5-Fu), gemcitabine (GEM), leucovorin, irinotecan, capecitabine, pembrolizumab, and platinum compounds as shown in Table 1.Of these, GEM is regarded as the agent of choice in pancreatic cancer management. [8,9]Although these regimens are widely used in the clinic, their relative efficacy remains substantially low.This is highly associated with several mechanisms of chemotherapeutic resistance including the high expression of multi-drug resistance (MDR) efflux pumps in the tumor cells, molecular and pathophysiological hindrances due to the pancreatic tumor microenvironment (TME), and their relatively short half-life due to extensive metabolism, all of which ultimately reduce intracellular concentration and thus, cytotoxic efficacy. [10]pecifically, the TME surrounding the pancreatic cancer tissue plays a crucial role in disease progression and poor treatment response. [14]The TME of pancreatic cancer is composed of a diverse array of cell populations, including normal and cancerassociated fibroblasts (CAFs), quiescent and activated pancreatic stellate cells (qPSCs, aPSCs), monocytes, regulatory T cells, tumor-associated macrophages, and myeloid-derived suppressor cells, as illustrated in Figure 1. [15]These cells play a crucial role in tissue stroma, which is responsible for maintaining homeostasis in the pancreatic environment.Under normal conditions, the extracellular matrix (ECM) preserves the proper function of cells by regulating their proliferation and movement, while preventing abnormal cell growth.However, in the case of pancreatic cancer, paracrine signaling and cross-talk between these cells and local pancreatic cancer cells have been shown to contribute to cancer cell survival and invasiveness. [14,16,17]This cellular cross-talk can also lead to stromal cell activation, resulting in the formation of dense fibrotic tissue via a process called desmoplasia. [16]This dense, collagen-rich layer surrounds the tumor and accounts for 70-90% of the tumor volume in both primary and metastatic PDAC stroma. [18]In this way, desmoplasia creates a physical barrier to blood vessel formation, and infiltration of immune cells into the tumor, whilst promoting tissue hypoxia associated with low blood perfusion, imparting redox imbalances, and increasing the overall acidic nature of the pancreatic TME.These adverse conditions collectively hamper the effective delivery of therapeutic drugs to the pancreatic cancer tissue. [19]igure 1.Schematic representation depicting the intricate pancreatic cancer TME, featuring prominent cell types, and paracrine signaling pathways.Highlighting the factors contributing to suboptimal drug delivery.
Among the stromal cells in pancreatic cancer, CAFs and their subsets including aPSCs are key components of the PDAC stroma. [14,15]These are activated by a variety of mediators including tumor necrosis factor  (TNF-), interleukins (IL-1, IL-6, and IL-10), platelet-derived growth factor, and transforming growth factor  (TGF-).Activated CAFs and PSCs then secrete their own mediators, contributing to the development of the desmoplastic layer through the production of collagen, fibronectin, glycosaminoglycans, proteoglycans, and laminin.[22][23][24] The intricate composition of the pancreatic TME has led to complex and challenging clinical outcomes in pancreatic cancer. [22,25]To develop effective therapeutic strategies, further exploration and understanding of the detailed dynamics of the TME are necessary. [26]In recent years, advanced smart materials, particularly those based on stimuli-responsive nanoparticles (NPs), have been explored for use in pancreatic cancer management.Stimuli-responsive nanoparticles are delivery systems that respond to both internal (pH, ROS, hypoxia, enzymes, and temperature) and external triggers (ultrasound, light, and magnetic fields), allowing for controlled and targeted drug release. [27]Ongoing research in this field offers promising prospects for innovative therapeutic and diagnostic approaches.This is because multiple functions can be engineered into these nanomaterials which can address the different aspects of the disease, ranging across tissue ingress, transport across cellular barriers, and delivery of diverse therapeutic agents particularly in pancreatic cancer.

Nanotechnology in Pancreatic Cancer
Medical nanotechnology utilizes materials in the nanometre size range, typically between 10 and 200 nm, as diagnostics, therapeutics, and carriers/supports for drugs and devices. [28]Lipid/RNA NPs have shown the effectiveness of medical nanomaterials during the ongoing SARS-CoV-2 pandemic, [29] and further development of NPs as drug delivery vehicles continue to address challenges associated with many therapeutic agents, such as poor aqueous solubility, unfavorable pharmacokinetics, toxicity concerns and limited stability. [30]The past two decades have witnessed significant progress in cancer pathology and nanoscience, resulting in the development of numerous nanomaterials for cancer treatment and diagnosis. [31]iven their distinct features, such as large surface area to volume ratio, availability in numerous morphologies and physical forms, and accessible surface functionality, synthetic nanomaterials are promising candidates for pancreatic cancer therapeutics. [32,33]36] Through careful design and modification, NP-based drugs can achieve superior specificity and bioavailability, lower normal tissue cytotoxicity, enhanced loading capacity, extended half-life, and controlled drug release patterns compared to conventional therapies. [37,38]oreover, these particles can be further tailored by coformulating multiple drugs within a single NP platform.This allows synchronization of the pharmacokinetics and biodistribution of each drug component, enabling their synergistic delivery to pancreatic tumors. [39]In a previous study by Liu et al., a poly(ethylene glycol)-b-poly(d,l lactide) (PEG-PLA) micelle formulation called PIM, which co-encapsulates paclitaxel (PTX) and itraconazole (ITA), was used in PDAC treatment.PIM improved the pharmacokinetics and tumor accumulation of PTX and ITA relative to the free drugs, leading to enhanced efficacy in inhibiting tumor growth.The micelle formulation inhibited the hedgehog (Hh) pathway and reduced cancer cell proliferation.In animal models, PIM demonstrated superior anti-cancer efficacy compared to PTX or ITA alone or their physical mixture.In addition, PIM significantly inhibited tumor growth and reduced Ki-67-positive cells, indicating suppressed proliferation. [40]ene therapy has also shown potential but faces implementation challenges, which can be overcome using drug delivery systems such as NP formulations. [41]In an attempt to advance this technology, an amphiphilic polyglutamate amine (APA) polymeric NP for delivering miR-34a and siRNA targeting PLK1 was developed.The APA nanocarrier effectively formed complexes with the small RNAs which were successfully internalized in pancreatic cancer cells.In vitro experiments confirmed that when combination treatment was delivered using the polyplexes there was a synergistic effect, with increased miR-34a levels, downregulation of target genes, and reduced cell viability, migration, and clonogenicity. [42]he wide variety of materials available for NP fabrication contributes to their ability to improve specificity and bioavailability, lower toxicity to normal tissues, increase drug loading capacity, prolong the duration of action, control drug release, co-encapsulate multiple drugs, and deliver genetic material.For example, natural phospholipids, and some synthetic nature-inspired lipids exhibit exceptional biocompatibility and biodegradability, enabling the assembly of liposomal colloidal vesicles with high drug-loading capacities that constitute a significant proportion of clinically approved nanomedicines. [43]In pancreatic cancer, liposomes have offered specific targeting flexibility for both insoluble and water-soluble compounds, enhancing drug delivery precision. [44]However, they face several drawbacks including low stability during long-term storage, high cost, limited payload, and rapid burst release. [45]Solid lipid nanoparticles (SLNs) were introduced as a stable alternative, composed of solid lipids or blends that exhibit slower release rates. [46]Yet, SLNs face challenges in encapsulating hydrophilic bioactive compounds due to their high-water content. [47]To overcome these limitations, nanostructured lipid carriers (NLCs) were developed.NLCs, consisting of a blend of solid and liquid lipids, offer higher loading capacity and controlled release rates in pancreatic cancer. [48,49]olymeric NPs, such as micelles and dendrimers, have also been used in pancreatic cancer therapy.Polymers can be designed to interact with certain drug compounds to increase drug loading and can be modified to achieve desirable release kinetics. [50]For example, the ratio of the component monomers of polylactic-co-glycolic acid (PLGA) can be adjusted to control the degradation rate of the particles. [51]Hydrophilic polymers, like poly(ethylene glycol) (PEG), can provide NPs with a "stealth" coating, effectively hiding them from immune recognition. [52]Some of these materials have already reached clinical trials, such as those mentioned in a study by Saif et al., which demonstrated the tolerability of micellar systems for paclitaxel delivery in pancreatic cancer patients, indicating their clinical potential. [53]However, challenges in precise synthesis, loading efficiency, and long-term biocompatibility exist.Rigorous evaluation of toxicity, scalability, and consistent quality in large-scale production is essential. [54]ybrid nanoparticles, specifically lipid-polymer hybrids, offer potential in the management of pancreatic cancer since they offer a versatile approach to drug delivery, combining the advantages of lipid and polymeric nanoparticles. [55]One of their significant advantages lies in their ability to deliver siRNA effectively, a feat demonstrated through cationic polymers interacting with siRNA and a lipid layer encapsulating and stabilizing the complex.These complexes have already been involved in codelivering chemotherapeutic agents like GEM and HIF1a siRNA (si-HIF1a) for pancreatic cancer treatment resulting in a substantial reduction in mRNA expression, indicating their potential in genetic therapies. [56]Hybrid lipid-albumin nanoparticles have also been developed and have been employed to overcome the challenges posed by dense tumor stroma in PDAC, allowing deep nanoparticle penetration into the tumor tissue [57] Inorganic nanoparticles have emerged as promising tools in the management of pancreatic cancer, offering enhanced imaging capabilities and unique therapeutic properties. [58]Nanomaterials such as carbon nanotubes (CNTs), quantum dots (QDs), and modified iron oxide nanoparticles possess distinctive features, including electrical and optical properties, enabling precise targeting of cancer cells and tissues. [59]For instance, modified iron oxide nanoparticles have shown specific targeting of pancreatic cancer cells, leading to increased drug uptake and enhanced apoptotic activity. [60,61]Gold nanoparticles have also demonstrated their effectiveness in delivering chemotherapeutic drugs and mi-croRNA inhibitors to pancreatic cancer cells, displaying reduced toxicity to healthy cells while enhancing anticancer effects. [62]owever, the translation of some inorganic nanoparticles from cancer research to clinical practice faces significant challenges, with one of the primary concerns being the potential long-term toxicity associated with their use. [63]Additionally, each type of inorganic nanoparticle comes with specific drawbacks; for example, QDs are known for their toxicity, gold nanoparticles entail high production costs, and CNTs are non-biodegradable. [63]Therefore, a careful evaluation of the cost-effectiveness of nanoparticle production for biomedical applications is essential, weighing the potential benefits against the production expenses. [63]he "enhanced permeability and retention" (EPR) effect has been considered as a passive targeting route for NPs to reach certain tumor environments.The EPR hypothesis suggests that NPs of an appropriate size can bypass the leaky vasculature surrounding tumor tissues in which growth and blood vessel formation are highly abnormal.Consequently, some NPs have been shown to remain within these tumors for an extended period (ranging from days to weeks) because of limited lymphatic drainage. [64]owever, this effect is significantly compromised in pancreatic cancer due to the hypovascularity and dense desmoplastic stroma of the TME. [65]Nevertheless, active targeting in pancreatic cancer is still feasible by utilizing the high surface-to-volume ratio of NPs which allows for their functionalization with various ligands, such as antibodies, aptamers, and peptides. [66,67]This approach enables selective association with specific receptors in the tumor sites and enhanced cellular uptake into tumor cells, contributing to greater efficacy in pancreatic cancer treatment compared to existing methods. [52,68]These materials, amongst others, possess versatile properties that enable a wide range of NP design and application possibilities.
It is now accepted, as described in a recent extensive review, that pancreatic cancer is multifaceted, and that further understanding is needed of tumor genesis, progression, and the ongoing evolution of treatments, in order to develop better nanomedicine theranostic agents. [69]While existing review articles have broadly discussed the role of nanomedicines in managing pancreatic cancer or responsive nanomaterials in cancer generally, [70][71][72][73][74][75][76][77][78][79] there has been a notable absence of research specifically focused on stimuli-responsive materials tailored for pancreatic cancer.Consequently, our study addresses the specific realm of stimuli-responsive nanomaterials and explores the intricate mechanisms through which they are activated in pancreatic cancer.Our discussion encompasses both internal and external stimuli-responsive NPs, precisely engineered to release therapeutic substances in response to specific stimuli.We define different advanced smart materials involved in the development of these NPs, with a particular emphasis on nanomaterials responsive to light, ultrasound, or magnetic fields, and intrinsic biological triggers such as pH, redox potential, hypoxia, enzymatic, and temperature within complex biological environments (Figure 2). [80] number of approaches for creating such stimuli-responsive NPs have been explored in recent years, with examples summarized in Table 2.We focused on the latest advances in this area, to highlight the possibilities and challenges in designing and optimizing novel nanomaterials for enhanced therapeutic efficacy.

Ultrasound-Responsive Nanomaterials
Ultrasound (US)-triggered vehicles have emerged as promising candidates for theranostic applications, [96] offering enhanced UScontrast, improved imaging and diagnosis, and the possibility for image-guided drug delivery, allowing for precise targeting and localization of therapeutic agents. [97]Furthermore, US-responsive carriers can achieve triggered drug release in response to ultrasonic stimulation, which can be remotely activated, providing spatiotemporal control over drug release at the tumor site. [98]his localized drug delivery minimizes off-target effects and reduces systemic toxicity. [99,100]In pancreatic cancer, US-responsive moieties have demonstrated enhanced permeability of several therapeutic and diagnostic agents across the thick desmoplastic stromal layer upon US stimulus, including chemotherapeutic drugs, genes, and imaging agents.Various types of carriers that respond to US stimulation have been investigated for their ability to deliver drugs in a controlled manner, including nanobubble-NP complexes, liposomes, micelles, and microbubbles.These carriers are known for their capacity to exploit cavitation effects, which represent the formation of tiny empty spaces filled with gas and vapor in the nearby tissue and their implosive collapse when subjected to sound waves, enabling them to respond to the US and facilitate drug delivery.These nanomaterials can be • Panc02 cell lines were used.
• US stimulation led to a decrease in cell viability.
• Zr-MOF group showed a viability decrease of 68%.
• Panc02 cell lines were subcutaneously injected into the right hind limb of Female BALB/C nude mice.
• Control, US, Zr-MOF@AIPH, and Zr-MOF + US groups did not show any therapeutic effect.

•
In the treatment group (Zr-MOF@AIPH + US), there was a significant inhibition of tumor growth, which was 41.9% higher than in the PBS group. [81] Hollow mesoporous silica NPs (HMSNs) l-arginine (LA).HMSN surfaces were modified with PEG and targeting peptides Ultrasound responsive • LA as the nitric oxide (NO) donor upon US stimulation.
• On the 30th day, the survival rate increased from 0% to 80%.
• This improvement in survival rate was observed compared to the control group. [82] Liposomes • PANC-1 cells were implanted into the flank of nude mice.

•
The in vitro cytotoxicity results indicated that P@-Gem-HSA-NPs exhibited the strongest cytotoxicity compared to other treatment groups.
• Tumour growth in mice was more effectively inhibited by the combination of P@-Gem-HSA-NPs with laser irradiation compared to free drug and NPs without irradiation.
[ • Treatment of cultured cells with hyaluronidase and collagenase increased the uptake of magnetic NPs.
• Hyaluronidase treatment resulted in a 28% increase in iron deposits per cell.
• As a result thermal doses increased by 15-23% compared to magnetic hyperthermia without enzyme treatment.
• Heat-induced cell death also increased as a consequence of enzyme treatment.

•
The findings indicate that hyaluronidase improves NP infiltration, affects thermal treatment, and leads to cell depletion, suggesting a positive impact on tumor growth inhibition.• BxPC-3 and Mia PaCa-2 cells were used.
• A combination of GEM encapsulated within PEG-DB and PEG-PY encapsulated GDC showed greater effectiveness than the same concentration combination of the free drugs.

•
The main findings indicate that in Mia PaCa-2 cells, both GEM and pSL showed similar IC50 values whilst HA-pSL was higher.
• wIn Gr2000 cells, both pSL and HA-pSL showed significantly higher IC50 values compared to free drug; free GEM (4447 Nm) >
• Mia PaCa-2 and resistant Gr2000 xenograft models were developed by injecting cell lines subcutaneously at the right flank of NOD Scid mice.

•
In Mia PaCa-2 models, HA-pSL exhibited a tumor weight improvement ratio of 6.4, while pSL had a ratio of 2.7.
• Similarly, in GEM-resistant pancreatic cancer (Gr2000 models), HA-pSL showed a tumor weight improvement ratio of 3.1, and pSL had a ratio of 2.1 compared to free drugs.
[91] (Continued) and HeLa cells were used to assess biocompatibility) • Results demonstrated biocompatibility and minimal cytotoxicity, even at higher concentrations of DG@Lips NPs.
• PANC 1 cells injected subcutaneously into the right forelimb area in nude mice.
• DG@Lips-Gels group with laser irradiation exhibited significant tumor suppression, with a relative tumor suppression rate of 91.73%. [94] Thermally sensitive micelles composed of an elastin-like polypeptide (ELP) were used.• After 7 days, significant tumor inhibition was observed.
• Treated mice showed a median survival of 27 days compared to 7 days for control. [95] Adv. Therap.102][103][104][105][106] Amongst the widely used US-directed NP methodologies is focused ultrasound (FUS). [99]FUS represents the delivery of ultrasound waves from an extracorporeal source to a selected localized target site.Its ability to induce both thermal and non-thermal effects suggests potential applications in cancer therapy. [107]This technology has already reached Phase I clinical trials in pancreatic cancer management under the name "PanDox". [108]The treatment consists of FUS to pre-warm the target region, followed by intravenous infusion of ThermoDox™, a thermoresponsive liposome encapsulating doxorubicin, while continuing FUS application.FUS is then repeatedly applied to the target site to release the drug.Important clinical limitations were noted, including the need for identifying accurate acoustic tissue properties to ensure adequate heating and effective drug release.The study emphasizes the current lack of human data and the need to use relevant information for treatment planning.Further evaluation is required to understand the impact of changes in frequency on tumor tissue.The study also emphasizes the importance of considering abdomen shape in treatment planning and the challenges posed by larger tumors.Respiratory motion is identified as a risk, necessitating respiratory control for uniform temperature distribution.The findings suggest the need for further exploration of FUS-responsive nanomaterials in clinical applications and raise important limitations.Nonetheless, this study presents data that supports the future administration of FUS in the management of pancreatic cancer. [108]nother promising strategy for pancreatic cancer therapy involves the use of targeted NPs that carry drugs, combined with the technique of ultrasound-mediated microbubble destruction (UTMD).This combination has shown remarkable improvements in the uptake of NPs by cancer cells in vitro and tumor retention in vivo. [109]UTMD is a non-invasive technique that combines ultrasound waves and microbubbles to enhance drug delivery and therapeutic efficacy. [110]Microbubbles, typically filled with an inert gas, are injected into the bloodstream and can be selectively activated by ultrasound waves at the desired target site.[112] This phenomenon, known as sonoporation, allows for improved drug penetration and accumulation in the tumor tissue (Figure 3a). [112]ive confocal microscopy with membrane-specific dyes allowed real-time tracking of sonoporation. [113]The research revealed synchronized membrane perforation and resealing upon ultrasound exposure.Successful resealing occurred for small pores, but large ones or those without calcium ions failed to close. [113]t a subcellular level, sonoporation disrupted the actin cytoskeleton, observed through real-time imaging, indicating potential long-term effects on cellular functionality. [113]In pancreatic cancer, UTMD has been shown to shift tumor-associated macrophage (TAM) polarization from an M2 to an M1-like phenotype, effectively impeding tumor growth and metastasis.Furthermore, UTMD exhibited the capacity to enhance tumor vessel normalization by increasing pericyte coverage and improving endothelial barrier integrity, ultimately enhancing drug efficacy and suppressing tumor spread. [114]This phenomenon was attributed to the interaction between microbubbles and With UTMD, microscopic bubbles containing an inactive gas in the bloodstream are specifically triggered using ultrasound waves at the pancreatic tumor site.When subjected to ultrasound, these tiny bubbles rapidly expand and contract, generating mechanical forces that temporarily disturb cell membranes and enhance the permeability of blood vessels and the stromal layer (sonoporation), allowing an easier uptake of NPs into the cancerous tissue.b) A representation of sonodynamic therapy in pancreatic cancer.The NPs are delivered to the TME where they are internalized by endosomes and colocalized within lysosomes containing degradative enzymes.The NPs are then lysed releasing the chemotherapeutic drug (purple) and sonosensitizer (red).The chemotherapeutic drug induces its cytotoxic activity whilst the sonosensitizer is activated by ultrasound through sonoluminescence or sonophysicochemical effects producing ROS which leads to DNA damage and cell death.
ultrasound, leading to enhanced drug delivery and membrane permeability through sonoporation.Combining the advantages of NPs with UTMD was also shown to increase the accumulation and penetration of NPs in tumors with low EPR levels, suggesting its potential for the improvement of pancreatic cancer treatment. [115] one study, researchers loaded paclitaxel (PTX) into functionalized organic NPs with anti-CA19-9 antibodies that specifically target pancreatic tumor cells. [116]The NPs were prepared using a methoxy poly(ethylene glycol)-polyl(actic-co-glycolic) acidpolylysine (mPEG-PLGA-PLL) triblock copolymer and UTMD was utilized to improve delivery to the irradiated tumor.The in vitro release studies clearly indicated a sustained release of PTX from the PTX-NPs-anti CA19-9 nanoparticles over a period of 3 days.This was in contrast to the rapid release of 100% free PTX within the initial 8 hours.It is worth noting, however, that the authors missed an opportunity to investigate the impact of UTMD on drug release, which could have added valuable insights to the study.In vitro experiments on Capan-1 cells showed that the combination of PTX-NPs-anti CA19-9 with UTMD resulted in a low IC50 value, significant cell cycle arrest at the G2/M phase, and a higher rate of apoptosis in the treated cells.In subcutaneous mouse pancreatic tumor models, the combination therapy of antibody-functionalized NPs and UTMD resulted in the highest accumulation of the therapeutic agent within the tumor cells, which induced the highest tumor inhibition rate compared to free PTX and PTX-NP-anti CA19-9 NPs without UTMD.Consequently, the mean survival times of tumor-bearing nude mice were improved. [116]n a different study, a novel approach was developed to tackle the dense ECM and hypovascular networks commonly found in solid pancreatic tumors.The researchers devised a biodegradable pH/redox responsive nanoplatform using hollow mesoporous organosilica NPs (HMON) as a potential delivery system for two drugs: GEM and pirfenidone (PFD), an anti-fibrosis medication.In vitro release studies of PFD from PFD@HMON-Gem nanoparticles were investigated under various pH conditions.The presence of the GEM capping effectively controlled PFD release, with less than 20% released at pH 7.4 over 8 hours.The release was significantly lower than uncapped PFD@HMON, which released over 70% at pH 7.4 in the same time frame.The drug release from PFD@HMON-Gem was pH-dependent, reaching 67% after 48 hours at pH 6.5, triggered by the cleavage of the acetal covalent bond between Gem and HMON particles.GSH further increased PFD release at both pH 7.4 and 6.5 due to the hydrolyzation of the disulfide bond within the framework.However again, no information about UTMD-induced drug release was provided.To further enhance the effectiveness of this technique, UTMD was employed, which disrupted the surrounding tissues and facilitated deeper particle penetration. [117]In vivo studies further supported the effectiveness of the delivery system, showing the accumulation of PFD@HMON-Gem NPs in tumor tissues, particularly with the aid of UTMD.The NPs also exhibited prolonged circulation time and down-regulation of ECM components within tumor tissues, surpassing the performance of the free drugs alone. [117]onodynamic therapy (SDT) has also emerged as a promising non-invasive approach for the treatment of pancreatic cancer. [118,119]In SDT, ultrasound waves are utilized to activate sonosensitizers, leading to the production of reactive oxygen species (ROS) that selectively target and destroy cancer cells (Figure 3b). [118,120]Several ROS generation mechanisms have been hypothesized and involve sonoluminescence, as well as sonophysicochemical effects. [121,122]Sonoluminescence is the process in which light is emitted by the excitation of gaseous species dissolved in solution through acoustic cavitation and the emitted light excites sonosensitizers, inducing the generation of ROS (including 1 O 2 and OH .). [123][124][125] Sonophysicochemical effects involve the direct production of ROS from sonosensitizers by the high pressure and temperature created through the implosion of ultrasound-produced cavitation gas bubbles following a thermal energy transfer mechanism. [122,126,127]Figure 4 illustrates the chemical structure of a number of these compounds. [128]he localized ROS production generated from sonosensitizers can induce oxidative stress, disrupt the tumor stroma, enhance drug penetration, and promote tumor cell death. [121,123]However, the dense stromal barriers surrounding pancreatic tumors contribute to hypoxic regions within the TME, which limits the efficacy of SDT.To address this challenge, researchers developed hollow TiO 2 NPs loaded with collagenase (Col-H-TiO 2 NPs) which have the ability to break down stromal barriers as well as generate ROS. [135]In a patient-derived xenograft (PDX) model, the administration of Col-H-TiO 2 NPs followed by ultrasonic irradiation resulted in enhanced drug release and increased intracellular ROS generation.It exhibited favorable biocompatibility, efficient cellular uptake, and improved penetration within tumor models.In vivo experiments showed tumor suppression, necrosis, and apoptosis in treated groups, particularly when combined with ultrasound.Collagen degradation and reduced tumor interstitial fluid pressure were observed, indicating enhanced antitumor potential and tumor permeability.Additionally, enhanced intratumoral ultrasound signals, which enable precise ultrasound imaging-guided and highly effective SDT for pancreatic cancer were shown.This proposed NP system offered the ability to remodel the TME and enhance visualization of pancreatic cancer when combined with SDT, which is particularly relevant for the management of pancreatic cancer patients. [135]itric oxide (NO) has been considered as a safe and effective technique for treating PDAC by enhancing drug penetration through modulation of the desmoplastic layer. [136]In one study, barium titanate (BaTiO 3 ) NPs were encapsulated into a US responsive nanoprodrug, CPT-thioketal-R-PEG2000 (CRB) and camptothecin (CPT) and the NO-donor l-arginine were conjugated to the polymer using a thioketal bond forming CPT-t-R-PEG2000@BaTiO 3 .The thioketal bond was broken when CRB NPs generated ROS in the hypoxic tumor environment upon US stimulation, thereby delivering NO and CPT to the tumor site simultaneously as demonstrated by in vitro and in vivo experiments.Consequently, the desmoplastic layer was diminished, leading to improved delivery and effectiveness of CPT.Specifically, in vivo studies revealed that the production of NO upon ultrasound irradiation significantly augmented the tumor penetration of CPT.This can be attributed to the ability of NO to diffuse freely into deep tumor sites, effectively modulating the ECM and thereby suppressing chemoresistance while improving the overall antitumor efficiency. [137]uture advancements in ultrasonic physics and sonochemistry are expected to improve the outcomes for pancreatic cancer patients through enhanced drug efficacy, reduced side effects, and personalized treatment strategies.

Near-Infrared Photo-Responsive Nanomaterials
Photo-responsive NPs have also shown potential for the treatment of pancreatic cancer. [84]These materials and the optical systems that activate them provide several advantages, including simple operation, the ability to achieve high spatiotemporal resolution of tumor targeting, precise control over wavelength and intensity, and non-invasiveness. [138]30][131][132][133][134] fundamental principles governing photo-responsive NPs mostly revolve around the encapsulation of photo-responsive agents called photosensitizers. [139]These agents respond to various wavelengths of light, inducing cytotoxic pharmacological effects on target cells. [140]When a photosensitizer absorbs a photon of light, it undergoes a transition from a ground state to a transiently excited singlet state which can follow two distinct pathways.First, it may emit fluorescence as it reverts back to the ground state-a property extensively employed in clinical applications for imaging and photodetection. [139,141]Alternatively, it can assume a relatively long-lasting reactive triplet state by flipping its excited electron through a process known as intersystem crossing.[144] Moreover, the triplet state can transfer its energy to molecular oxygen, resulting in the formation of a singlet oxygen which may also induce cellular toxicity and tumor ablation. [144]hile various photosensitizers can react to different wavelengths of light, the specific use of ultraviolet (UV)-visible light responsive drug delivery systems in pancreatic cancer is limited due to the substantial phototoxicity caused by UV light and the shallow tissue penetration depth of both UV and visible light in the pancreas. [138,145]In contrast, near-infrared (NIR) light (650-950 nm) and near-infrared two-zone (NIR-II) (1,000-1,700 nm) exhibit minimal phototoxicity and possess deeper tissue penetration capabilities, which is appropriate for management of pan-creatic cancer as shown in Figure 5b. [146,147]NIR light can be employed to activate different photosensitizers by employing appropriate optical materials as transducers.These photosensitizers, in turn, generate ROS for photodynamic therapy (PDT) or heat for photothermal therapy (PTT). [148,149]Figure 4 illustrates the chemical structures of a number of these compounds. [132]For example, the anti-tumor effect of a novel NIR agent, organic heptamethine carbocyanine dye (DZ), conjugated to HMG-CoA inhibitor simvastatin (SIM) (DZ-SIM) on pancreatic cancer cell lines was investigated.The results showed that DZ-SIM significantly decreased the survival of PDAC cells, including Mia PaCa-2, UN-KPC960-Luc, and BxPC3.When combined with GEM, the NIR dye and irradiation caused a greater cytotoxic effect on pancreatic cancer cells relative to monotherapy.DZ-SIM also decreased colony formation and inhibited PDAC cell migration, and was shown to accumulate in tumor tissues but not in normal tissues.DZ-SIM was shown to disrupt mitochondrial function, resulting in reduced bioenergy production in cancer cells.Studies using PDAC mouse models also demonstrated the effectiveness of DZ-SIM in inhibiting tumor growth and metastasis. [150]As a result, the incorporation of components sensitive to ROS, thermal effects, and short-wavelength light into drug delivery systems might improve targeted therapy significantly for pancreatic cancer.(Figure 5c). [148,149]ombination NIR/cytotoxic delivery systems have included PTX-loaded PLGA microspheres, with gold NPs (GNPs) conjugated through polydopamine (pD) linkers, for pancreatic cancer management (GNPs-pD-PTX-PLGA). [152]The microspheres demonstrated three times greater cytotoxicity in PANC-1 cell Once internalized by the cells, these NPs release their chemotherapeutic agent which initiates its cytotoxic effects, while the photosensitizer becomes activated upon exposure to near-infrared (NIR) light, transitioning from the ground state to a singlet excited state.This singlet excited state can either return to the ground state, generating fluorescence suitable for clinical imaging or undergo intersystem crossing to form a long-lasting triplet excited state.This triplet state can interact with substrates or oxygen molecules within the TME, generating ROS that may induce cytotoxic activity.b) Penetration depths of different wavelengths of light across a tissue show that NIR light has the best penetration depth through soft tissue. [151]c) An illustration of how NIR is used in the management of pancreatic cancer.A photosensitizer loaded within an NP system is injected into the tumor tissue.Upon NP accumulation at the tumor site, the tumor is exposed to NIR light, inducing the production of heat and/or ROS leading to the selective ablation of the cancerous tissue.
lines after NIR treatment compared to the group that did not receive NIR treatment.Additionally, the generation of ROS increased, resulting in augmented apoptosis and photothermal effects induced by NIR.Consequently, the expression levels of antioxidant enzymes were downregulated. [152]In another study, Doxorubicin (DOX) loaded liposomes containing the IRsensitizer dye ICG were coated with the extracted cell membrane of SW1990 pancreatic cancer cells.These particles demonstrated controlled release of DOX in response to light stimulation and exhibited remarkable efficacy in both in vitro and in vivo models of pancreatic cancer.Upon irradiation with near-infrared light (808 nm), the liposomes released DOX at the tumor site and generated photodynamic effects attributed to ICG which elicited robust anti-tumor activity.In addition, given the surface structural similarities between the NPs and the SW1990 cells, a homologous targeted delivery to pancreatic tumor tissues was achieved, resulting in heightened NIR-II fluorescence imaging intensity. [153]These findings highlight the synergistic chemo-photothermal therapy potential of similar NPs in the management of pancreatic cancer.
Photo-responsive nanomaterials were also used for the development of photothermal immunoassay kits for the sensitive monitoring and detection of CA 19-9, a tumor marker used for the detection of pancreatic cancer.Here, a 3D-printed device was integrated with a digital thermometer and a microplate coated with antibodies for detection. [154]CaCO 3 microspheres were encapsulated with Prussian Blue NPs (PBNPs) which served as both signal amplifier photo-heat conversion materials and labels for antibody detection.The PBNPs induced a change in temperature in the detection solution by converting NIR light (808 nm laser irradiation) to heat.The photothermal immunoassay exhibited high sensitivity, precision, and specificity, and the particles also exhibited good storage stability despite their compositional complexity.The assay exhibited a linear detection range spanning from 1.0 U mL −1 to 100 U mL −1 L, with a detection limit of 0.83 U mL −1 L for the tumor marker CA 19-9.The methodology demonstrated advantages such as an experimentally facile synthesis of the PBNP-CaCO 3 photothermal materials, simple activation via NIR light irradiation, and potential via the 3D-printing technique to be applied for measuring biomarkers other than CA 19-9. [154]lthough NIR-responsive NPs have shown promising results in pre-clinical studies, there is a significant concern regarding the efficiency of NIR in penetrating deep within the pancreatic tumor tissue in a clinical setting.The dosing of NIR and its method of administration needs to be optimized to match human physiological conditions to enable successful clinical translation.Injected magnetic NPs surfaces decorated with chemotherapeutic agents are precisely guided and concentrated at the pancreatic tumor through the application of a static external magnetic field.This targeted approach allows for precise localization of the NPs at the pancreatic tumor site.Subsequently, magnetic hyperthermia is generated through the interaction between alternating magnetic fields (AMF) and the NPs, enabling the release of these molecules at the target site.This property not only facilitates targeted drug delivery but can also be employed to induce a relatively selective thermal ablation of the tumor tissue leading to cancer cell death.
Addressing this critical aspect remains a research challenge for future safer and more effective drug delivery strategies.

Magnetic-Responsive Nanomaterials
[157] These particles have been engineered using diverse materials, such as transition metals (Fe, Ni, Co), metal oxides (Fe 2 O 3 ), metal alloys (Fe─Ni, Fe─Pt, Fe─Co, Co─Pt), and rare metals (Gd, Eu) to enhance their application performance and improve physiological outcomes. [158,159]By applying a static external magnetic field, these NPs can be precisely guided and concentrated at the tumor site, enhancing drug delivery to cancer cells while minimizing exposure to healthy tissues.Drug molecules can be attached to the surface of the magnetic NPs, allowing for increased drug accumulation in the desired location following magnetic field guidance (Figure 6).This targeted delivery system has been investigated for various applications, including localized drug release at tumor sites for pancreatic cancer treatment. [160]ron oxide is the most commonly-used magnetic material for cancer diagnosis and treatment applications, primarily due to its authorization for clinical use from the FDA. [161]Iron oxide-based NPs exhibit good magnetic properties and demonstrate mostly superior stability against degradation with lower toxicity profiles compared to other magnetic particles. [162,163]Magnetic iron oxide NPs with diameters between 15 to 100 nm have shown efficiency in many biological applications. [164]For instance, Huang et al. developed a precisely targeted pancreatic cancer treatment using coated magnetic iron oxide NPs coated with milk-protein (casein). [165]In this study, the ironoxide-based NPs included amino-terminal fragments (ATF) of urokinase plasminogen activator for tumor-targeting and the antitumor drug cisplatin.The NPs demonstrated specific binding to uPAR-overexpressing pancreatic cancer cells and exhibited enhanced cytotoxicity compared to free cisplatin in vitro.In an orthotopic pancreatic tumor model, the functionalized NPs exhibited superior antitumor activity, resulting in reduced tumor weight and fewer metastases compared to controls.The NPs also showed improved safety profiles, with reduced toxicity observed in liver and spleen tissues.MRI studies confirmed the accumulation of ATF-functionalized NPs at the tumor site, and ex vivo measurements further validated the higher accumulation of these particles compared to non-targeted therapy.Prussian blue staining and immunofluorescence labeling confirmed the co-localization of targeted NPs with uPAR-expressing tumor areas.The treatment also led to a decrease in collagen bundles, suggesting disruption of the TME and improved drug delivery. [165]agnetic NPs can also be utilized in drug delivery for other purposes, such as magnetic hyperthermia. [166]This technique exploits the ability of certain magnetic materials to generate heat when exposed to an alternating magnetic field (AMF), which can be used to destroy cancer cells or trigger the release of drugs from an NP carrier (Figure 6). [167]Recently, magnetoliposomes (ML), comprising superparamagnetic iron oxide NPs (SPION) enveloped by a phospholipid bilayer, were developed for magnetic fluid hyperthermia (MFH) in both 2D pancreatic cancer cell cultures and 3D organoids. [168][171] In the context of 2D cell cultures (Mia PaCa-2 and PANC-1), four MFH configurations were examined, including no ML, intracellular ML, intra-and extracellular ML, and extracellular ML.Notably, solely extracellular MFH exhibited pronounced cytotoxicity, leading to an immediate reduction in cell viability in both cell lines following treatment, although PANC-1 cells showed increased viability 24 hours post AMF exposure.Clonogenic assays demonstrated that intracellular MFH lowered clonogenic potential by 23% in both cell lines, while extracellular MFH induced substantial reductions.Importantly, when applied to organoids, ML exhibited negligible cytotoxicity in the absence of hyperthermia.Morphological analysis suggested that ML adhered to the organoid surface without internalization, and subsequent molecular investigations revealed no significant alterations in proliferation or apoptosis markers.Finally, MFH treatment utilizing extracellular ML resulted in a marked reduction in organoid viability, showcasing its potential for PDAC treatment. [168]imilarly, iron oxide-based magnetic NPs were developed and assessed in both in vitro 3D collagen-based Mia PaCa 2 cell line models and in vivo tumor xenograft models. [172]To render these NPs water-stable and traceable, a poly(maleic anhydridealt-1-octadecene) (PMAO) coating modified with a fluorophore (TAMRA) was applied, followed by functionalization with glucose to enhance stability in biological environments and cellular uptake.The exposure of these 3D models to AMF under various conditions revealed a notable increase in magnetic NPs internalization.In in vivo experiments involving murine models, AMF exposure subsequent to magnetic NPs injection resulted in heightened magnetic NPs penetration into tumor regions, likely attributable to heat-induced alterations in the extracellular matrix.This could significantly enhance drug delivery in tumors characterized by dense stromal tissue.Additionally, exposure to magnetic NPs induced a heat-triggered immune response; however, variations in treatment effectiveness were observed among subgroups of animals.Animals demonstrating a more favorable response to MH treatment exhibited minimal magnetic NPs leakage into the liver and spleen, while those with a weaker response displayed detectable magnetic NPs accumulation in these organs.These findings underscore the critical importance of precise magnetic NPs biodistribution within the tumor for treatment efficacy. [172]he combination of magnetic hyperthermia and chemotherapy in a single magnetic NP system was also explored (Figure 6).In one study, a GEM delivery system for pancreatic cancer was described using magnetic NPs with a maghemite core coated with the polysaccharide dextran (MNP-GEM). [173]The NPs exhibited a high capacity for heat generation, making them suitable for mag-netic hyperthermia applications.In cell viability studies, MNP-GEM demonstrated a significant cytotoxic effect, particularly in GEM-resistant pancreatic cancer cells.The nanocarrier was less toxic to non-tumoral cells, highlighting its selectivity toward cancer cells.Internalization studies indicated enhanced uptake of MNP-GEM in pancreatic cancer cell lines.The enhanced cytotoxic effect was attributed to increased ROS production in certain cell lines.Cell death mechanism studies revealed differences between GEM and MNP-GEM, possibly due to the release mechanism of the drug from the nanocarrier.Combination studies with magnetic hyperthermia demonstrated enhanced cytotoxicity of MNP-GEM when exposed to an alternating magnetic field (AMF). [173]n a different study, mechanisms by which magnetic hyperthermia of superparamagnetic iron oxide NPs (MF66) induced pancreatic cancer cellular death were explored.In vitro experiments demonstrated that magnetic hyperthermia treatment resulted in cell necrosis and elevated ROS production.In vivo studies using PANC-1 subcutaneous xenograft models revealed that magnetic hyperthermia treatment effectively suppressed tumor growth and decreased the expression of proliferation markers.Interestingly, the presence of magnetic NPs alone in cancer cells was found to induce ROS formation, triggering apoptosis.This phenomenon was attributed to the presence and subsequent degradation of magnetic NPs within the cells, further contributing to ROS production. [174]hile the use of magnetic NPs in drug delivery holds great promise, it is crucial to note that further studies and clinical trials are necessary to fully understand their potential benefits, safety, and long-term effects. [175]Clinical trials and regulatory approvals play a vital role in translating this innovative approach into effective treatments for pancreatic cancer patients.

pH-Responsive Nanomaterials
The acidic TME in pancreatic cancer results in a significant increase in tumor cell proliferation, migration, and invasion capabilities. [176]The lower extracellular pH (pH 6.5-6.8)observed is primarily attributed to the heightened production of lactic acid through anaerobic glycolysis, a consequence of the tumors' elevated metabolic rate and inadequate clearance of lactic acid and carbon dioxide, among other mechanisms. [177]Endosomal pH has been shown to be even lower (pH 5.0-5.5). [178,179]184][185] In many cases, these are bound to the NP shell using pHresponsive linkers, some of which are mentioned in Figure 4. [131] Various studies have focused on developing pH-responsive NPs for targeted drug delivery in pancreatic cancer.One such study by Ray et al. utilized PEG-containing amphiphilic polycarbonate block copolymers with tertiary amine side chains to create a pH-responsive drug delivery system. [186]This approach Figure 7.The distinctive tumor microenvironment (TME) in pancreatic cancer, characterized by acidity, high levels of ROS/glutathione (GSH), and hypoxia, offers an opportunity for targeted drug delivery.In the acidic TME (pH 6.5-6.8) and even more so within endosomes (pH 5.0-5.5),pH-responsive NPs can be tailored with pH-sensitive linkers, facilitating controlled drug release at specific pH levels within the tumor.Similarly, redox-responsive NPs leverage the abundant ROS/GSH environment in the tumor to selectively cleave disulfide bonds, releasing therapeutic drugs.Hypoxia-responsive NPs are designed to respond to the low-oxygen regions of the TME (pO 2 between 0-5.3 mmHg), leading to NP collapse and drug release.These responsive mechanisms are minimally triggered under normal physiological conditions, ensuring precise and smart drug release at the tumor site.
aimed to co-deliver an ERK inhibitor (ERKi) and GEM.The pHresponsive NPs exhibited selective drug release behavior, with over 80% of ERKi and 85% of GEM released at pH 4.5 after 24 hours.In vitro cytotoxicity assays demonstrated that the encapsulated drugs in the pH-responsive NPs had lower IC50 values compared to free ERKi and GEM.Further evaluation in tumorbearing mice revealed that the combination of pH-responsive NPs loaded with ERKi and GEM significantly impeded tumor progression and induced tumor regression through suppression of cell proliferation.pH-responsive NP combination therapy showed enhanced efficacy compared to free drug combination treatment.Additionally, the pH-responsive NP combination effectively suppressed ERK activity and exhibited a favorable safety profile without inducing hepatotoxicity or renal toxicity. [186]n another study, Fan et al. developed a pH-responsive NP system composed solely of membrane-disruptive macromolecules for treating pancreatic cancer. [181]In this study, acid-responsive materials were derived from copolymers of hexylmethacrylate, dimethyl aminoethylmethacrylate, and methacrylic acid, denoted as Poly-14K and Poly-35K, and showed pH-sensitive disruption of red blood cell membranes.When formulated into micelles, described as M-14K and M-35K NPs, acid-activatable, membrane-disruptive behavior was observed.M-14K demonstrated acid-activated cytotoxicity on cancer-associated fibroblasts, selectively eliminating them under acidic conditions.Further experiments using fluorescence and electron microscopies confirmed the ability of M-14K to permeabilize mammalian cell membranes.In comparison, the chemotherapy drug GEM showed pH-insensitive cytotoxicity, indicating off-target effects.In vivo studies in xenograft pancreatic BxPC-3 tumors demonstrated that both M-14K and M-35K had long circulation times and preferentially accumulated in tumor tissues.Importantly, mice treated with M-14K at pH 6.8 showed significant suppression of tumor growth, highlighting the effectiveness of its acidactivated cytotoxicity.Notably, the NPs effectively permeated the dense pancreatic tumor tissue, while significantly reducing the expression of ECM components and activated cancer-associated fibroblasts. [181] pH-sensitive polymer, mPEG-G4-DnPEA, was also synthesized for the delivery of a GEM pro drug (Pro-Gem) to pancreatic cancer tissue.[187] By modifying G4 PAMAM dendrimer with different feed ratios of N,N-dipentylethylamine (DnPEA), and mPEG, polymers with varying sensitivity to pH changes were prepared which exhibited ultrahigh pH-responsiveness, with transition pH shifting from 6.4 to 6.8 as DnPEA graft numbers decreased.The selected PEG-G4-DnPEA30 polymer, with a transition pH of 6.8, was designated as tumor pHsensitive NPs (SPN) and Pro-Gem was successfully loaded into SPN, with significant size reduction observed at pH 6.8.In contrast, pH-insensitive NPs (IPN) were prepared as a control.Both SPN@Pro-Gem and IPN@Pro-Gem demonstrated enhanced drug release under acidic conditions, with SPN exhibiting a higher release rate.In vitro studies showed that SPN facilitated cell internalization and induced greater cell apoptosis compared to IPN.Further investigations in 3D multicellular spheroid models supported the superior penetration and efficacy of SPN.
In vivo experiments demonstrated that SPN@Pro-Gem effectively inhibited tumor growth and modulated the tumor immune microenvironment, suggesting its potential as a promising chemoimmunotherapy approach for pancreatic cancer. [187]H-responsive NPs have also been used for targeted delivery of small interfering RNA (siRNA), a promising approach for silencing specific genes involved in pancreatic cancer.PDAC is known for its dense ECM that hinders CD8+ T cell infiltration and limits drug penetration.Wang et al. delivered TGF- receptor inhibitors (LY2157299) and siRNA targeting PD-L1 (siPD-L1) pH-responsive clustered NPs which inhibited the TGF- pathway and the PD-L1 checkpoint. [188]The study proposed a strategy where the designed NPs responded to the acidic extracellular pH of the tumor, enabling the release of therapeutic agents and promoting intratumoral T-cell infiltration.The delivered LY2157299 effectively inhibited the activation of pancreatic stellate cells (PSCs) and reduced collagen levels, while siPD-L1 promoted penetration into the tumor and suppressed PD-L1 gene expression.In both subcutaneous and orthotopic tumor models, the clustered NPs exhibited synergistic effects, leading to significant tumor growth suppression.The NPs also enhanced siRNA penetration into multicellular spheroids and showed higher cellular uptake, resulting in effective gene silencing.Furthermore, the NPs promoted the infiltration of cytotoxic CD8+ T cells and induced the secretion of interferon-, indicating the activation of antitumor immune responses.Additionally, the NPs effectively inhibited the TGF- signaling pathway and diminished PSCinduced collagen deposition.This type of combination therapy using pH-responsive NPs demonstrated superior antitumor efficacy compared to monotherapy, highlighting the potential of this approach in pancreatic cancer treatment. [188]ipid-based pH-responsive NPs have also shown promise in pancreatic cancer treatment.Tang et al. developed hyaluronic acid (HA) functionalized pH-sensitive liposomes (HA-pSL) for intracellular drug delivery to overcome GEM resistance in pancreatic cancer. [91]The study investigated two types of liposomes: pH-sensitive liposomes (pSL) and hyaluronic acidgrafted liposomes (HA-pSL), both carrying GEM as the cargo.These liposomes demonstrated responsiveness to changes in pH, releasing the drug rapidly under acidic conditions.The pH-responsive component in the liposomes was 1,2-dioleoylsn-glycero-3-phosphoethanolamine.In vitro studies using Mia PaCa-2 cells and GEM-resistant Gr2000 cells revealed the effectiveness of the liposomes.Mia PaCa-2 cells showed similar IC 50 values for GEM and pSL, with HA-pSL exhibiting a higher IC 50 value, while in Gr2000 cells, both pSL and HA-pSL had significantly higher IC 50 values compared to free GEM, with HA-pSL demonstrating the lowest IC 50 value.In vivo studies using Mia PaCa-2 and Gr2000 xenograft models further supported the efficacy of the liposomes.HA-pSL demonstrated a higher tumor weight improvement ratio compared to pSL and free GEM in both models, indicating superior in vivo activity. [91]ccordingly, pH-responsive NPs offer a promising approach for treating pancreatic cancer by selectively releasing therapeutic agents in response to the acidic TME, overcoming challenges such as dense ECM and chemoresistance.However, it is noteworthy to mention that these studies have been performed against in vitro cell lines and mouse models which may not reflect the true pH found within the TME in human pancreatic cancer tissue.Thus, further research investigating this technology in a clinical setting is needed to demonstrate whether a similar effect will be observed in human patients.The NP design can then be further adjusted to enhance drug delivery efficiency and explore combination therapies in pancreatic cancer treatment.

Redox-Responsive Nanomaterials
[191] This heightened ROS level plays a multifaceted role in driving the disease by promoting cell proliferation, facilitating metabolic adaptation, enhancing angiogenesis, and activating PSCs within the desmoplastic TME, consequently promoting tumor invasion and bolstering pancreatic cancer cell growth and survival. [191,192]195] Notably, GSH, recognized as the most abundant cellular antioxidant, assumes a pivotal role in ROS regulation by directly neutralizing them through the donation of reducing equivalents.[198] As such, redox-responsive NPs have been exploited for the targeted drug delivery in pancreatic cancer by harnessing the intracellular and extracellular imbalances of ROS and GSH, achieving controlled drug release, improving therapeutic efficacy and minimizing off-target effects (Figure 7). [199,200]In particular, the use of disulfide bonds in the NP structure quickly became a common approach for redox-responsive NP system formulation as this functionality is cleaved in the presence of high GSH levels. [201]Xu et al. have utilized this technology by developing a redox-responsive thiolated gelatin-based NP system for targeted delivery of therapeutic agents to pancreatic cancer cells with efficient payload release. [202]The system enabled site-specific delivery of wild-type p53 expressing plasmid DNA and GEM, targeting the overexpressed epidermal growth factor receptor (EGFR) in pancreatic cancer cells.In vivo studies in mice demonstrated that the targeted NPs exhibited improved targeting efficiency, leading to increased transfection efficiency and suppression of tumor growth.The NPs induced apoptotic pathways, as evidenced by increased mRNA expression of proapoptotic transcription factors and protein levels of apoptotic biomarkers.Furthermore, the thiolated gelatin NPs loaded with wt-p53 exhibited a notable efficiency in transfection and an enhanced production of p53 protein.This led to the activation of apoptotic pathways downstream.GEM conjugated to gelatin NPs exhibited enhanced cytotoxicity compared to the free drug.The combination treatment using targeted gelatin NPs resulted in synergistic tumor growth inhibition and increased apoptotic activity. [202]In another study, a redoxsensitive micellar delivery system with GEM covalently bound to polymer chains using disulfide bonds was developed.The micelles showed a high loading capacity of 12% (w/w) GEM and exhibited a 2.5-fold higher accumulation of GEM in an orthotopic pancreatic cancer model compared to free GEM.To achieve selective delivery of GEM and the miRNA (miR-519c), the micelles were further modified with a GSH-sensitive polypeptide and an EGFR-targeting peptide (GE11).In vitro and in vivo studies demonstrated that the mixed micelles inhibited Hh signaling, tumor cell invasion, and HIF-1, effectively inhibiting pancreatic tumor growth.The concurrent use of miR-519c and GEM demonstrated a synergistic impact on the eradication of cancer cells and effectively restored the sensitivity of GEM-resistant pancreatic cancer cells to GEM treatment.The micelles also demonstrated stability and enhanced cellular uptake of miR-519c. [203]edox-responsive NPs have also shown promise as a targeted drug delivery approach with the ability to minimize the lack of selectivity of current nuclear localization peptides (NLS) in pancreatic cancer. [134]NLS are short peptide sequences found in specific proteins that aid in their transportation into the cell nucleus for carrying out specialized functions.Anajafi et al. introduced an innovative approach utilizing a 'masked' NLS peptide that is selectively activated by the overexpression of matrix metalloproteinase-7 (MMP-7) enzyme within the pancreatic TME. [134]By attaching the vesicles to a protected NLS, they observed that MMP-7 hydrolyzed the masking peptide, thereby exposing the NLS on the surface of the polymersomes.This mechanism facilitated the delivery of cargo, including doxorubicin and curcumin, directly to the nucleus of pancreatic cancer cells.The drug-loaded polymeric vesicles were assessed in 3D spheroids of pancreatic cancer and normal cells.The results demonstrated a preferential cytotoxic effect on cancer cells compared to normal cells, indicating enhanced tumor selectivity when compared to the free drug. [134]Other illustrations of redox-responsive polymers are mentioned in Figure 4. [134,203] Overall, redox-responsive NPs hold significant potential for targeted delivery of therapeutic agents in pancreatic cancer.However, similarly to pH-responsive NPs, since many of these redoxresponsive formulations have not been reported in human clinical trials, further testing of this technology in a clinical setting is required to demonstrate whether a similar effect will be observed.

Hypoxia-Responsive Nanomaterials
Hypoxia, a lack of oxygen in the body tissues, presents as a characteristic feature of pancreatic tumors and plays a crucial role in its highly aggressive nature and resistance to conventional treatments. [204]Numerous and severe hypoxic regions within the pancreatic tumor are found with a marked median tissue partial oxygen pressure (pO 2 ) ranging from 0 to 5.3 mmHg (0 to 0.7%), in contrast to the adjacent healthy pancreas which exhibits a pO 2 of 24.3 to 92.7 mmHg (3.2 to 12.3%). [205]The hypoxic microenvironment observed primarily stems from the presence of desmoplastic fibrotic stroma, rapid cancer cell proliferation, and inadequate vascularization, which contribute to increased oxygen consumption and compromised oxygen supply. [205,206]ypoxia-responsive NPs, which share many features with the wider field of redox-responsive materials, are designed to specifically target and deliver therapeutic agents to hypoxic regions within pancreatic tumors. [205]They are typically engineered to respond to the low oxygen environment by undergoing rapid structural destabilization and therefore releasing loaded drugs or therapeutic agents in a controlled manner (Figure 7).Specifically, nitroimidazole, nitrobenzyl alcohol, and azo derivative linkers have been used owing to their reduction chemistries applicable to the hypoxic nature surrounding the TME (Figure 4). [133]his targeted approach allows for more effective treatment that delivers therapeutic payloads directly to the tumor site and overcomes the limitations of traditional chemotherapy. [207]Research on hypoxia-responsive NPs for pancreatic cancer treatment is ongoing and has shown promising results in preclinical studies.Recently, Chen et.al. formulated an aptamer-functionalized hypoxia-responsive ultra-small NP, namely dendri-graft polylysine (DGL), co-loaded with STAT3 inhibitor HJC0152 and GEM (s(DGL)n@Apt). [208]The hypoxia-responsive NPs possessed the ability to alter their surface charge within the TME and undergo a size reduction in response to hypoxia.This induced the release of ultra-small DGL particles carrying GEM monophosphate, which effectively penetrated deep into the tumor and promoted endocytosis of chemotherapy drugs, thereby enhancing their efficacy.In vivo results demonstrated that tumor samples from the s(DGL)n@Apt group displayed the lowest Ki-67 signal intensity, indicating reduced cell proliferation, and the strongest cleaved caspase-3 signal intensity, suggesting enhanced cell apoptosis.Concurrently, the STAT3 inhibitor HJC0152 was able to inhibit the overactivated STAT3 signaling pathway in both tumor cells and tumor stroma.This modulated the stroma barrier but also re-educated the TME, transforming it into an immune-activated state.
Kulkarni et al. designed a polymer-based hypoxia-responsive nanoformulation of PEG-azobenzene-poly(lactic acid) (PLA) NPs encapsulated with GEM and erlotinib, an EGFR inhibitor. [209]nder hypoxic conditions, 90% of the encapsulated cargo was released in 50 minutes due to the breakdown of the particles through the cleavage of the azobenzene linker, demonstrating hypoxic triggered drug release.Cellular viability did not change under normal conditions when 3D BxPC-3 spheroids where exposed to the hypoxia-responsive polymersomes.This was not the case in hypoxic conditions, where the spheroids showed a decrease in cell viability (24% ± 4%) when treated with the drug-loaded polymersomes, relative to free drugs (48% ± 2%), indicating superior efficacy of the drug-containing hypoxiaresponsive polymersomes. [209]ollowing these results, the same research group functionalized similar polymersomes with iRGD peptide and loaded GEM and napabucasin (Nap) (a STAT3 inhibitor) to improve the selectivity of these particles in pancreatic cancer. [210]In vivo studies in mice revealed significant reductions in tumor size when treated with peptide-functionalized GEM, Nap, and combination NPs.Specifically, the tumor size was reduced by 5.1, 6.5, and 7.5 folds, respectively, compared to the control group that received no drugs.In addition, growth suppression was observed in the free drug groups, with a reduction in size of 1.6 fold for both free GEM and free Nap, although these reductions were not statistically significant compared to the control.An interesting finding was that the tumors treated with iRGD exhibited a distinct property change, becoming physically softer compared to the solid and dense control tumors.Upon resection, the tumors revealed sunken fluid-filled cores, indicating a substantial increase in tumor necrosis.These results suggest that these NPs could be multi-functionalized achieving greater targeting activity on tumor cells.
The development of hypoxia-responsive NPs is still in its early stages and there are several challenges that need to be addressed to treat pancreatic cancer such as the stability of the NPs in blood circulation, the specificity of the NPs to hypoxic regions, and the toxicity of the NPs.

Enzyme Responsive Nanomaterials
The overexpression of enzymes, particularly cathepsins and matrix metalloproteinases (MMPs), in pancreatic cancer has been extensively documented, underscoring their intricate involvement in tumorigenesis. [211]Alongside their established roles in ECM degradation and cancer progression, recent research has emphasized their vital contributions to fundamental processes like tumor formation, angiogenesis, and invasion. [211,212]Interestingly, pancreatic cancer patients consistently demonstrate elevated cathepsin expression, evidenced by a significant proportion of primary pancreatic tumors exhibiting positive staining for cathepsin B and L. [213] Consequently, there is a growing interest in the utilization of nanomaterials with enzymeresponsive cleavable bonds, particularly peptides with specific protease responsibility, which are commonly considered candidates for constructing nanoparticles with enzyme-responsive linkers. [214]This approach involves systemic injection of nanoparticles that remain inactive until they reach the tumor site, where increased enzymatic activity triggers drug release. [215]The objective is to minimize systemic side effects while optimizing the accumulation of therapeutic agents within the tumor, presenting a promising strategy for targeted pancreatic cancer treatment.
In one study, cadmium selenide (CdSe) /zinc sulfide (ZnS) core-shell quantum dots (QDs) were employed as vectors for GEM delivery, benefiting from their potent fluorescent properties, to investigate the activity of dual cathepsin B and MMP-9 enzymatic reaction-assisted drug delivery system. [216]The hydrophobic QDs were transformed into carboxylated QDs (c-QDs) by coating them with an amphiphilic polymer.Dual-enzyme-sensitive QDs (d-QDs) were prepared by a one-pot amide reaction of carboxylated QDs with GEM modified with GFLG-NH2, an amine terminal cathepsin B responsive linker (GEM-GFLG-NH2), PEGylated polymer with an MMP-9 responsive linker (PEG-GGPLGVRGK-NH2), and tumor-targeting motif which binds to integrin receptors on the surface of cancer cells called CycloRGD (c(RGD)fk). [217]These d-QDs, designed to respond to both MMP-9 and cathepsin B, exhibited stability in phosphate-buffered saline and demonstrated efficient cellular internalization in human pancreatic cancer (BxPC-3) cells.MMP-9mediated deshielding of the PEG shell exposed the targeting ligand RGD, enhancing cell uptake.Cathepsin B-sensitive cleavage of GEM from d-QDs was confirmed through HPLC analysis.In vivo experiments confirmed MMP-9-triggered enhanced cancer cell uptake and cathepsin B-mediated GEM release within tumor tissues.The dual-enzyme-sensitive GEM vectors exhibited enhanced therapeutic efficacy in BxPC-3 tumor-bearing nude mice compared to free GEM, with minimal side effects.Histological analysis and immunohistochemistry further supported the superior antitumor effect of the dual-enzyme-sensitive vectors, highlighting their potential as a promising strategy for targeted pancreatic cancer therapy. [216]n an RAFT (Reversible Addition-Fragmentation Chain Transfer) polymerization study, GEM-conjugated polymers were synthesized using GEM-methacrylate monomers with ester and amide linkers. [218]The resulting NPs demonstrated controlled drug release behavior under different pH conditions and in the presence of the enzyme cathepsin B. In a pH 7 environment, 100% availability of free GEM was observed after 1 h, however, only 2% of GEM was released after 1 day from the NPs, increasing to 20% after 30 days.Conversely, at pH 5, mimicking the intracellular acidic environment, 4% of GEM was released after 1 day from the NPs, escalating to 50% after 30 days due to accelerated hydrolysis of ester and amide linkages.The presence of Cathepsin B at pH 5 significantly enhanced GEM release from the NPs, with ≈10 and 70% released after 1 and 30 days, respectively, confirming the susceptibility of the GEM-polymer conjugate to enzymatic degradation, even without a specific peptide linker.Additionally, these NPs exhibited sustained cytotoxicity against Mia PaCa-2 pancreatic cancer cells for up to 30 days, surpassing the efficacy of free GEM. [218]imilarly, an advanced enzyme-activated GEM delivery system using nanodiamonds (NDs) to enhance pancreatic cancer chemotherapy was developed. [219]The method involved coating raw NDs with positive-charged polyethyleneimine (PEI) and negative-charged polyacrylic acid (PAA) bilayers for increased stability and enhanced surface carboxyl groups.PEG was introduced to prevent protein adsorption, ensuring prolonged circulation and tumor accumulation.GEM was linked to NDs via the GFLG peptide, enabling cathepsin B-induced controlled release.In vitro experiments revealed that GEM-bound NDs (ND-PEI-PAA-PEG-GEM) exhibited a 2.5 times slower drug release rate compared to free NH2-GFLG-GEM, possibly due to PEG interference with the enzyme's binding.Assays in BxPC-3 pancreatic cancer cells demonstrated comparable efficacy between NDbound GEM and free GEM, with minimal cytotoxicity from blank carrier NDs.In vivo experiments using BxPC-3 tumor-bearing nude mice showed ND-bound GEM's superior performance in inhibiting pancreatic tumor growth compared to free GEM, The current standard of care involves administration of free drugs through the IV route which is subjected to systemic drug distribution, extensive hepatic metabolism, and major side effect profile.Poor intracellular uptake of these drugs is also observed due to the hypervascularization of the tissue, thick desmoplastic stromal layer, and mutation in drug uptake mechanisms and efflux pumps of cancer cells.b) Overcoming the challenges of chemotherapy in pancreatic cancer using thermo-responsive micellar-based hydrogels.These polymers exist in a liquid state which undergoes a rapid sol-gel transition at body temperature leading to a sustained drug release at the tumor site.Since these formulations are administered intratumorally, they minimize systemic drug distribution and side effects whilst ensuring minimal hepatic metabolism.They also bypass the vascular route and thick desmoplastic layer allowing for a greater amount of drug at the tumor site.In addition, loading the chemotherapeutic drugs in NPs may further sustain the release and enhance drug uptake mechanisms whilst shielding the drug from degradation.
emphasizing the potential of ND-based drug delivery systems for advancing pancreatic cancer treatments. [219]hese studies reveal the promising potential of enzymeresponsive nanomaterials in managing pancreatic cancer.However, two key limitations must be considered.[222] Deeper insights into the spatial and temporal enzymatic activity patterns are crucial for precise delivery.Second, related enzyme families often share substrates, necessitating specific designs to enhance delivery effectiveness and prevent adverse reactions.Further research in pancreatic cancer enzymatic biochemistry is essential for developing clinically relevant delivery systems.

Thermosensitive Nanomaterials: Micellar Hydrogels
Responsive micellar materials offer the possibility to deliver drugs over an extended period while minimizing the adminis-tered dosage, reducing side effects in non-target tissues, and enhancing the therapeutic effectiveness at the site of the pancreatic tumor (Figure 8a). [94]225][226] Upon in situ injection, the hydrogels release the drug payload in a controlled and targeted manner. [227,228]A significant advantage of micellar-based thermosensitive hydrogels is their noncytotoxic gelation process, eliminating the need for potentially harmful crosslinking agents or reactions, [229] and making them particularly suitable for pancreatic cancer treatment.Furthermore, the temperature differential between the tumor site and the formulation storage temperature allows for the instant gelation of temperature-responsive polymer hydrogels, which exhibit 3D networks with the ability to absorb and retain large volumes of water in their swollen state, providing a biomimetic environment specifically tailored for administration into pancreatic tumor tissue. [230]Figure 4 illustrates the structures of several polymers currently used in the development of thermoresponsive hydrogels. [129]The simplicity of triggering gelation and swelling behaviors in response to temperature changes makes thermosensitive hydrogels ideal as injectable drug delivery carriers for localized administration, minimizing invasiveness and improving treatment outcomes for pancreatic cancer patients.
In this context, Shi et al. introduced an injectable hydrogel that forms in situ, utilizing thermo-sensitive poly(d,l-lactide)-b-PEG-b-poly(d,l-lactide) copolymer micelles.This hydrogel was designed for the localized co-delivery of GEM and cisplatin as a combined therapeutic approach specifically for pancreatic cancer treatment.The composite hydrogel, loaded with two drugs, exhibited a distinct and reversible sol-gel transition from room temperature to physiological temperature.This characteristic enabled easy injection and facilitated the formation of a drug reservoir precisely within the targeted tumor area.In vitro drug release studies confirmed the sustained release of both drugs simultaneously with 91.8% and 59.7% of GEM and cisplatin, respectively being released over 10 days.The dual drug-loaded hydrogel system showed improved anti-tumor efficacy relative to the free drugs, as well as the single drug-loaded hydrogels in vitro.In vivo experiments on pancreatic cancer xenograft models demonstrated sustained, controlled, and localized drug release.The GEM-cisplatin/hydrogel exhibited significant tumor growth inhibition and achieved desirable synergistic therapy.The hydrogel also showed minimal systemic side effects in treated mice. [228]n another study, a thermosensitive micellar-based PLGA-PEG-PLGA polymer system hydrogel was developed and loaded with both GEM and rapamycin (RAPA), the latter serving as an mTOR signaling inhibitor. [231]The selected GEM:RAPA ratio (11:1) exhibited a synergistic effect and achieved a high encapsulation efficiency (>75%) within the thermoresponsive gel.The resulting gel particles demonstrated consistent size distribution, with particle sizes below 100 nm and a low PDI (<0.3).Notably, this mixed polymer system underwent a phase change near body temperature, offering potential advantages for hyperthermiabased treatments.Both in vitro and in vivo studies provided evidence of a synergistic effect between the drugs and controlled drug release from the gel, with 86% and 9.08% of GEM and RAPA, respectively being released over 168 hrs, resulting in enhanced therapeutic efficacy.Importantly, the system displayed reduced toxicity and prolonged drug residence within the peritoneal cavity, suggesting its potential for topical chemotherapy in PDAC treatment, with the potential to minimize adverse effects and improve patient outcomes.However, further research is necessary to gain a deeper understanding of the underlying mechanisms and tissue distribution in vivo. [231]anobiohybrid hydrogels, which combine inorganic NPs and biodegradable polymeric hydrogels, were also developed to address the limitations of GEM in pancreatic cancer treatment, including its short half-life, systemic toxicity, and the need for frequent administration.The injectable nanobiohybrid hydrogel was prepared by incorporating GEM into montmorillonite NPs, which were dispersed into a biodegradable and temperature-sensitive poly(-caprolactone-co-lactide)b-PEG-b-poly(-caprolactone-co-lactide) hydrogel.The surface charge of the MMT NPs became negatively charged when partially substituted with magnesium ions.The interaction between GEM and MMT NPs resulted in a reduction in the negative charge of the NPs.The nanobiohybrid hydrogel exhibited improved properties, including sol-gel phase transition behavior through improved mechanical stability, reduced the burst release of GEM at initial time points in in vitro release tests, where 40% and 12% of GEM was released from the pristine hydrogel and the nanobiohybrid hydrogel, respectively, over the first 12 h.In vivo studies on rats showed that the nanobiohybrid hydrogel retained its shape and degraded slowly over time demonstrating low cytotoxicity, making it suitable for biomedical applications.In a mouse model of pancreatic cancer, the GEM-loaded nanobiohybrid hydrogel exhibited superior tumor growth inhibition compared to saline and GEM solutions.No significant weight loss or side effects were observed in the treated mice. [232]urthermore, Shabana et al. developed a thermosensitive and biodegradable hydrogel that contained targeted NPs.This hydrogel was designed to achieve localized and sustained delivery of an NP system encapsulating GEM and PTX specifically to pancreatic cancer sites.The NPs were decorated with a fibronectin-mimetic peptide, PR_b, which interacts with overexpressed integrin 51 in pancreatic cancer.These PR_b-functionalized liposomes were encapsulated within a poly(-valerolactone-co-d,l-lactide)-b-PEGb-poly(-valerolactone-co-d,l-lactide) pentablock copolymer hydrogel, enabling controlled release of both drugs over time and overcoming limitations associated with systemic toxicity and repeated administration.The PR_b-functionalized liposomes showed selective internalization into pancreatic cancer cells, enabling optimal delivery of the two drugs.The hydrogel-liposomes combination exhibited an extended-release of GEM and PTX compared to free drugs within the hydrogel.Specifically, 92 ± 3% of GEM and 56 ± 2% of PTX were released over 6 days for free drugs in hydrogels.In contrast, it took 15 days for 91 ± 7% of GEM and 50 ± 7% of PTX to be released from liposomes in hydrogels.Additionally, the hybrid system of hydrogel and NPs exhibited remarkable effectiveness in eradicating human pancreatic cancer cells (PANC-1) and substantially impeded the growth of PANC-1 tumor spheroids.These results were achieved when compared to hydrogels containing non-targeted liposomes loaded with GEM and PTX, as well as free drugs, after one week of treatment. [227]imilarly, researchers developed a thermosensitive chitosan/N-[(2-hydroxy-3-trimethylammonium) propyl] chitosan chloride (HTCC)/glycerophosphate hydrogel system (CHG) loaded with liposomes encapsulating IRF5 mRNA/CCL5 siRNA (LPR).The liposomal/hydrogel thermoresponsive formulation (LPR@CHG) was used for targeted and sustained delivery of LPR NP complexes to pancreatic tumors. [233]The LPR@CHG hydrogel demonstrated physicochemical stability both in vitro and in vivo, including slow degradation and controlled release of LPR NPs, where 90.8% of NPs were released over 16 days.This system efficiently delivered mRNA and siRNA, resulting in the upregulation of IRF5 and downregulation of CCL5 expression.It also promoted the polarization of macrophages to the M1 phenotype and showed significant antitumor effects in vivo, inhibiting tumor recurrence and influencing the TME positively.Moreover, the hydrogel increased CD3+ T cell infiltration into the tumor.This research highlights the potential of RNA-based gene therapy combined with hydrogel delivery systems for cancer immunotherapy, offering a promising avenue for future studies and mRNA tumor vaccine development. [233]n conclusion, temperature-responsive hydrogels hold significant promise for improving drug delivery and treatment outcomes in pancreatic cancer.Their ability to undergo controlled phase transitions in response to temperature changes allows for targeted drug release and enhanced penetration within the TME.However, it is important to characterize these gels in clinical settings, primarily to ensure the safety of these materials as there are concerns over their robustness and whether 'dosedumping' may be of high risk upon application, which evidently may cause severe therapeutic toxicities. [234]Further research and clinical application of these systems are imperative for progressing the therapeutic outcomes of pancreatic cancer.

Multi-Stimuli Responsive Nanomaterials
The combination of multiple stimuli-responsive elements in drug delivery systems can significantly improve the therapeutic outcomes in pancreatic cancer management.In this context, Hao et al. developed pH/redox-responsive polyprodrug NPs (SPNs) through the electrostatic interaction of two types of block copolymers: PDMA-b-P(AEMA-DA) and t-P(CPTMco-DTPA(Gd)-co-CS)-b-PGEMA. [235]The PDMA-b-P(AEMA-DA) copolymer was prepared by polymerizing N,N-dimethyl acrylamide (DMA) and 2-aminoethyl methacrylamide hydrochloride (AEMA), followed by amidation to introduce acid-labile -carboxylic-acid amides.The t-P(CPTM-co-DTPA(Gd)-co-CS)b-PGEMA copolymer was based on CPT prodrug moieties, CS fluorophore, diethylenetriaminepentaacetic acid-gadolinium (DTPA(Gd)) MRI contrast agent, and methacrylamide) (PGEMA).These copolymers self-assembled in PBS buffer to form SPNs, which exhibited excellent stability and underwent size reduction and charge reversal in tumor acidic conditions.The degradation of SPNs released smaller polyprodrug NPs with positive charges, promoting deep tumor penetration.Furthermore, the release of the CPT prodrug from SPNs was triggered by reductive conditions, while the Gd moieties in the SPNs enhanced the magnetic resonance signals in a pHdependent manner.Cellular internalization studies confirmed the efficient uptake of SPNs by tumor cells at acidic pH, leading to enhanced cytotoxicity.In contrast, the acid-insensitive control NPs showed limited cellular uptake and lower cytotoxicity.In vivo studies also demonstrated that SPNs exhibited superior tumor penetration and showed promising results in delivering therapeutic drugs to deep-seated tumor cells with significant tumor inhibition and prolonged tumor retention. [235]n another study, PTT-responsive NPs were prepared using temperature-sensitive liposomes (DG@Lips). [94]The NPs consisted of semiconducting polymers Poly([2,5-bis(2decyltetradecyl)-2,5-dihydropyrrolo [3,4- [1,2,5]thiadiazolo [3,4f ]benzotriazole-4,8-diyl]) (DPP-BTz) and the chemotherapy drug GEM wrapped with amphiphilic polymers to obtain spherical NPs with a size c. 100 nm.The DG@Lips NPs exhibited NIR-II absorption, and their photothermal performance was concentration-dependent.The NPs showed photothermal stability and high photothermal conversion efficiency (PCE).Upon laser irradiation, the DG@Lips NPs released GEM due to the heat-sensitive responsiveness of the liposomes.Poly(d,llactide)-b-PEG-b-poly(d,l-lactide) copolymer hydrogels loaded with DG@Lips were prepared and showed reversible sol-gel transitions upon heating and cooling (DG@Lips-Gels).The hydrogel system exhibited sustained release of DG@Lips in a simulated tumor acidic microenvironment.In vitro experiments demonstrated the biocompatibility of DG@Lips NPs and the antitumor efficacy of the DG@Lips-Gels hydrogel system, combining PTT and chemotherapy.In vivo studies in pancreatic cancer tumor models showed that DG@Lips-Gels with laser irradiation effectively suppressed tumor growth. [94]imilarly, CTh (2,3,3-trimethyl-5-(thiophen-2-yl)-3H-indole) was synthesized as a photothermal therapy (PTT) compound designed for acid-activated absorbance. [236]CTh, when combined with GEM, was encapsulated within lipid NPs (C-G NPs).In vitro experiments assessing the PCE of CTh NPs revealed their ability to reach temperatures exceeding 75 °C under specific conditions while maintaining thermal stability when exposed to NIR laser irradiation.The calculated PCE for CTh NPs was 53.14% under physiological conditions (pH = 7.4) and increased to 56.76% under acidic conditions (pH = 6.0).Additionally, GEM release from the C-G NPs solution showed a slightly higher rate under NIR laser irradiation in acidic conditions, with release percentages of 70% and 82% recorded at pH 7.0 and pH 6.0, respectively, indicating their pH responsiveness.In vitro cell studies on PANC-1 cells illustrated the superior photothermal effects of C-G NPs, resulting in enhanced cellular uptake and significant cytotoxicity upon NIR laser exposure compared to conditions without irradiation.Furthermore, the study explored the impact of PTT on pancreatic stellate cells (PSCs) within the TME, revealing its potential to suppress the secretion of transforming growth factor-beta 1 (TGF-1) by PANC-1 cells and inhibit the activation of PSCs, ultimately enhancing drug permeability within the tumor.In vivo experiments, using a metastatic xenograft PDAC mouse model, corroborated the effectiveness of PTT in disrupting the dense ECM, thus facilitating drug penetration into solid tumors.The combined approach of PTT and GEM resulted in a substantial reduction in tumor volume without significant toxicity to vital tissues.PTT further influenced the TME by reducing collagen content and ECM components while suppressing the expression of TGF-1, alpha-smooth muscle actin (-SMA), and collagen I in tumor tissues. [236]hoto/thermo/pH-responsive NPs were also prepared for the delivery of DOX to pancreatic cancer tissue. [237]Here, porous polypyrrole NPs (PPy NPs) were synthesized through a templateguided chemical oxidation method, where octylamine (OTA) and polyvinylpyrrolidone (PVP) formed spherical micelles as templates for the subsequent PPy polymerization.These PPy NPs exhibited surface pores favorable for loading lauric acid (LA) through hydrophobic interactions.The carboxyl groups of LA were then activated, and bovine serum albumin (BL) was grafted onto the PPy NP surface to form stable PLB NPs.Characterization confirmed the successful modification and the resulting PLB NPs exhibited good colloidal stability.Furthermore, PPy NPs demonstrated efficient loading of the anticancer drug doxorubicin (DOX), with loading efficiency influenced by DOX concentration and PPy NPs concentration.The pH and temperatureresponsive drug release properties of PPy NPs were observed, with enhanced release at lower pH and elevated temperature.Additionally, PLB NPs displayed excellent photothermal conversion ability and maintained enzymatic activity for BL.Hemocompatibility and in vitro biocompatibility of PLB NPs were confirmed, with no observable chronic toxicological effects in vivo.A) Schematic representation of the preparation of sNP@G/IR, which possesses a core-shell arrangement that is dual responsive.B) The growth of bacteria-colonized tumors is effectively suppressed by sNP@G/IR, which eliminates intracellular bacteria within the tumor and ensures targeted drug delivery.This process comprises four stages: i) HAase in the extracellular matrix (ECM) aids in the separation of the shell, leading to the exposure of core nanoparticles (NP@G/IR); ii) the exposed NP@G/IR, possessing a diminished particle size and reversed charge, facilitates effective penetration into the tumor, effectively eliminating extracellular bacteria; iii) the rapid uptake of NP@G/IR triggers the removal of intracellular bacteria and enables the controlled drug release; iv) the stimulation of CD8+ T cell generation leads to the activation of the immune system.Reproduced with permission from Advanced Materials. [241]Copyright 2022, John Wiley and Sons. [202]B NPs exhibited efficient in vitro cancer cell therapy through combined photothermal and chemotherapy approaches.In vivo experiments demonstrated the synergistic anti-tumor effect of PLB NPs, especially when combined with photothermal therapy.Moreover, PLB NPs demonstrated photoacoustic (PA) imaging capabilities, enhancing tumor-specific imaging. [237] redox-responsive polymer-cisplatin conjugate, branched Polyethyleneimine-SS-cisplatin (BPEI-SS-Pt), was also synthesized with a high cisplatin loading of up to 33.4%. [238]y mixing BPEI-SS-Pt with HAase and amorphous calcium phosphate, pH/redox responsive nano-complexes (BPEI-SS-Pt/HAase@CaP) were formed.In vitro release experiments revealed that BPEI-SS-Pt/HAase@CaP demonstrated pH-sensitive rupture and enhanced release of HAase, with rapid platinum release in the presence of high GSH levels.In vitro evaluations in Panc02 cells demonstrated effective antitumor performance for both BPEI-SS-Pt and BPEI-SS-Pt/HAase@CaP, with dose-dependent responses.Cellular uptake studies revealed that the particles were uptaken mainly through clathrin-mediated endocytosis.In vivo studies using Panc02 tumor-bearing mice revealed that BPEI-SS-Pt/HAase@CaP exhibited superior antitumor efficacy compared to control groups and reduced systemic toxicity.TUNEL, Caspase-3, and Ki67 assays indicated enhanced apoptosis and reduced tumor proliferation in the treatment group.Moreover, BPEI-SS-Pt-5-FAM/HAase@CaP co-loaded with HAase effectively degraded hyaluronan in the tumor stroma, facilitating deeper drug penetration and enhanced chemotherapy efficacy in pancreatic cancer. [238]rug inactivation plays a crucial role in determining the outcome of a specific treatment.[241] A potential approach to combat drug inactivation is by delivering anticancer drugs along with specific antibiotics.This combination can effectively eradicate bacteria, overcoming drug resistance caused by bacterial activity. [241]Kang et al. synthesized and characterized SGPs (synthetic guanidine-based polymers) with PHMG (polyhexamethyleneguanidine) and redox-responsive moieties as shown in Figure 9a. [241]These polymers exhibited antibacterial activity, biocompatibility, and the ability to form NPs loaded with the anticancer drug GEM and the photothermal agent IR1048.The NPs were coated with HA to enhance tumor targeting.The resulting NPs demonstrated stability, controlled release of GEM, and the ability to generate photothermal response and they effectively eradicated bacteria when combined with laser irradiation by disrupting bacterial cell membranes and walls.In vitro studies showed rapid internalization of the NPs through CD44 receptor recognition and successful lysosomal escape (Figure 9b).When tested on 3D tumor spheroids, the NPs penetrated deep into the tumor tissues, especially after pretreatment with hyaluronidases.In vivo experiments with mice confirmed that the NPs accumulated in the tumor region exhibited superior targeting capabilities and induced a significant temperature increase in tumors, demonstrating good photothermal performance.The NPs were found to be biocompatible and safe with no adverse effects on the mice.In tumor-bearing mice, the NPs showed excellent therapeutic efficacy, suppressing tumors and achieving a tumor inhibition rate of 98.7% when combined with photothermal therapy.Additionally, they effectively eliminated bacteria within tumors, reduced tumor volume, and increased the survival time of mice.The NPs also stimulated an immune response, activating dendritic cells and CD8+ T cell infiltration in the tumor, indicating their potential for immunotherapy (Figure 9b). [241]

Clinical Studies Using Nanotechnology For Management Of Pancreatic Cancer
Numerous clinical trials have investigated the potential utilization of NPs in treating pancreatic cancer.Two of the notable developments were the approval by the FDA in 2013 of Abraxane, a formulation that comprises solvent-free albumin-bound PTX NPs, and in 2015 of Onivyde, a liposomal injection of Irinotecan, to be used for treating pancreatic cancer patients.This was found to yield significantly improved clinical outcomes.Since then, many clinical trials have investigated the combination of these nano-formulations with free chemotherapeutic agents including GEM, Capecitabine, 5-Fu, Leucovorin, Bemcetinib, and platinum compounds (NCT03278015, NCT04371224, NCT04796948, NCT02562716, NCT03649321).Other novel formulations are currently being investigated for their potential clinical activity in pancreatic cancer as shown in Table 3.It is imperative to emphasize that a considerable number of these trials pertain to non-responsive NPs, rather than stimuli-responsive nanomaterials.This further reinforces our previous assertions regarding the necessity to examine the effects of these particles within clinical environments, in order to evaluate their likely patient impact.

Conclusion and Future Perspectives
The current systems for drug delivery in pancreatic cancer have shown poor therapeutic outcomes due to various challenges associated with the TME.The structural heterogeneity of the biological targets and limited accessibility of target cells caused by factors such as the dense desmoplastic stromal layer and compromised endothelial blood vessels, contribute to these challenges.Furthermore, the enhanced permeability and retention effect observed in other types of cancer does not translate effectively to pancreatic cancer.
To address these issues, the development of nanocarriers sensitive to exogenous or endogenous stimuli offers a promising solution for targeted drug delivery in pancreatic cancer.In this review, we have focused on advanced material nanoplatforms that respond to specific external stimuli such as ultrasound, near-infrared, and magnetic therapy, as well as stimuli within the TME, including low pH, high GSH concentration, hy-poxia, temperature difference, and excessive ROS.These stimuliresponsive systems provide opportunities for precise drug release at specific locations and times, using diverse materials and architectures.This approach leads to enhanced anti-tumor efficacy and reduced toxicity by specifically targeting tumor tissues.
Modulating the stroma surrounding the pancreatic cancer cells, whether induced by the stimuli-responsive nanomaterials or the introduction of stroma-targeted and vascular-modifiable agents into the nanocarriers, also holds great potential in the management of pancreatic cancer.This combination therapy could potentially overcome several biological hindrances that make it difficult for conventional therapy to penetrate through the TME. [242]Examples include a sequential treatment that combines GEM-loaded magnetic NPs with metformin, which induces stromal depletion to facilitate the delivery of GEM and dramatically reduce tumor burden. [243]Metformin activated the AMPK pathway and reduced TGF- expression, inhibiting stromal synthesis whilst disrupting the dense stroma and improving the penetration of GEM-loaded magnetic NPs in a pancreatic cancer model. [243]Other studies involved the use of NPs to modulate the PSCs, degrade the ECM, regulate the tumor vasculature, and improve the immune response surrounding the pancreatic cancer site. [244]he utilization of multi-stimuli responsive drug delivery systems in pancreatic cancer also holds significant promise for improving selectivity and cytotoxicity specifically on tumor cells.
The key lies in the strategic design of platforms capable of responding to both the internal TME of pancreatic cancer and external stimuli, thereby triggering a controlled release of therapeutic agents.This approach offers the potential to achieve heightened selectivity and increased cytotoxicity against cancerous cells.
While many stimuli-responsive systems have shown promise in in vitro and in vivo preclinical models, only a few have progressed to the clinical stage.Challenges related to the complexity and scalability of architectural design, batch-to-batch variation, poor stability, and production costs hinder the translation of these systems from the laboratory to practical clinical use.Simplified and innovative fabrication methods are needed to ensure costeffectiveness, reproducibility, stability, and large-scale production with quality control.
The efficacy, safety profile, and long-term side effects of these systems also require further investigation and remain subjects of debate due to their novelty.Although therapeutic effects have been reported in pancreatic cancer-bearing mouse models, these findings may not accurately predict their safety and efficacy in clinical trials since the mouse models of pancreatic TME may not fully reflect the human TME.Establishing preclinical animal models that closely mimic the human TME, including patient-derived tumor models, organoids, complex 3D coculture spheroids, organ-on-chip systems, and other relevant models, is crucial for evaluating the clinical potential of stimuli-responsive NPs.
Moreover, the toxicity of these NPs is multifaceted and depends on factors such as composition, physicochemical properties, and dosage.The benefit-to-risk ratio must be carefully evaluated based on the intended medical application.Strategies for assessing the short-term and long-term biosafety of stimuli-responsive NPs should include the use of metabolizable or biodegradable nanomaterials and comprehensive studies on drug distribution, pharmacokinetics, pharmacodynamics, blood biochemistry, hematology, and long-term toxicological evaluation.
Additionally, careful consideration of the cancer stage, whether localized or metastasized, becomes pivotal in determining the optimal route of administration, be it intravenous or intratumoral.Notably, novel techniques for drug administration in pancreatic cancer have emerged, such as Endoscopic Ultrasound-Guided Fine Needle Injection therapies encompassing immunotherapy, chemotherapy, and biological therapies, which have demonstrated minimal adverse events and promising efficacy in treating pancreatic cancer. [245]The integration of these stimuli-responsive nanomaterials with such administration techniques holds the potential to further augment the cytotoxic activity and selectivity of these particles.
In conclusion, stimuli-responsive nanocarriers hold great promise for the treatment of cancer, including pancreatic cancer.Further advancements in research can lead to the development of multifunctional nano-delivery systems for practical clinical applications, making significant contributions to human health in the future.

•
Orthotopic model was developed with BxPc3-luc2 injected into the pancreas.

Figure 2 .
Figure 2. Schematic representation of the application of both external and internal stimuli responsive 'smart' nanomaterials in the management of pancreatic cancer.

Figure 3 .
Figure 3.An illustration representing different techniques for utilizing ultrasound-responsive smart nanomaterials for the management of pancreatic cancer.a) Schematic representation of NP accumulation assisted by UTMD in pancreatic cancer.With UTMD, microscopic bubbles containing an inactive gas in the bloodstream are specifically triggered using ultrasound waves at the pancreatic tumor site.When subjected to ultrasound, these tiny bubbles rapidly expand and contract, generating mechanical forces that temporarily disturb cell membranes and enhance the permeability of blood vessels and the stromal layer (sonoporation), allowing an easier uptake of NPs into the cancerous tissue.b) A representation of sonodynamic therapy in pancreatic cancer.The NPs are delivered to the TME where they are internalized by endosomes and colocalized within lysosomes containing degradative enzymes.The NPs are then lysed releasing the chemotherapeutic drug (purple) and sonosensitizer (red).The chemotherapeutic drug induces its cytotoxic activity whilst the sonosensitizer is activated by ultrasound through sonoluminescence or sonophysicochemical effects producing ROS which leads to DNA damage and cell death.

Figure 5 .
Figure 5. a) Illustration depicting the multifaceted mechanism of action of theranostic dual-loaded NPs containing both photosensitizer and chemotherapeutic agents on pancreatic cancer cells.Once internalized by the cells, these NPs release their chemotherapeutic agent which initiates its cytotoxic effects, while the photosensitizer becomes activated upon exposure to near-infrared (NIR) light, transitioning from the ground state to a singlet excited state.This singlet excited state can either return to the ground state, generating fluorescence suitable for clinical imaging or undergo intersystem crossing to form a long-lasting triplet excited state.This triplet state can interact with substrates or oxygen molecules within the TME, generating ROS that may induce cytotoxic activity.b) Penetration depths of different wavelengths of light across a tissue show that NIR light has the best penetration depth through soft tissue.[151]c) An illustration of how NIR is used in the management of pancreatic cancer.A photosensitizer loaded within an NP system is injected into the tumor tissue.Upon NP accumulation at the tumor site, the tumor is exposed to NIR light, inducing the production of heat and/or ROS leading to the selective ablation of the cancerous tissue.

Figure 6 .
Figure 6.An illustration of magnetic responsive NP therapy in pancreatic cancer.Injected magnetic NPs surfaces decorated with chemotherapeutic agents are precisely guided and concentrated at the pancreatic tumor through the application of a static external magnetic field.This targeted approach allows for precise localization of the NPs at the pancreatic tumor site.Subsequently, magnetic hyperthermia is generated through the interaction between alternating magnetic fields (AMF) and the NPs, enabling the release of these molecules at the target site.This property not only facilitates targeted drug delivery but can also be employed to induce a relatively selective thermal ablation of the tumor tissue leading to cancer cell death.

Figure 8 .
Figure 8. a) The current limitations of chemotherapy in the management of Pancreatic cancer.The current standard of care involves administration of free drugs through the IV route which is subjected to systemic drug distribution, extensive hepatic metabolism, and major side effect profile.Poor intracellular uptake of these drugs is also observed due to the hypervascularization of the tissue, thick desmoplastic stromal layer, and mutation in drug uptake mechanisms and efflux pumps of cancer cells.b) Overcoming the challenges of chemotherapy in pancreatic cancer using thermo-responsive micellar-based hydrogels.These polymers exist in a liquid state which undergoes a rapid sol-gel transition at body temperature leading to a sustained drug release at the tumor site.Since these formulations are administered intratumorally, they minimize systemic drug distribution and side effects whilst ensuring minimal hepatic metabolism.They also bypass the vascular route and thick desmoplastic layer allowing for a greater amount of drug at the tumor site.In addition, loading the chemotherapeutic drugs in NPs may further sustain the release and enhance drug uptake mechanisms whilst shielding the drug from degradation.

Figure 9 .
Figure9.A) Schematic representation of the preparation of sNP@G/IR, which possesses a core-shell arrangement that is dual responsive.B) The growth of bacteria-colonized tumors is effectively suppressed by sNP@G/IR, which eliminates intracellular bacteria within the tumor and ensures targeted drug delivery.This process comprises four stages: i) HAase in the extracellular matrix (ECM) aids in the separation of the shell, leading to the exposure of core nanoparticles (NP@G/IR); ii) the exposed NP@G/IR, possessing a diminished particle size and reversed charge, facilitates effective penetration into the tumor, effectively eliminating extracellular bacteria; iii) the rapid uptake of NP@G/IR triggers the removal of intracellular bacteria and enables the controlled drug release; iv) the stimulation of CD8+ T cell generation leads to the activation of the immune system.Reproduced with permission from Advanced Materials.[241]Copyright 2022, John Wiley and Sons.[202]

Cara Moloney -
received her Ph.D. in BioNano Interactions from University College Dublin under the supervision of Dermot F. Brougham.Her current research focuses on the development of nanoparticle and hydrogel drug delivery systems for the systemic and local delivery of chemotherapeutics, and the assessment of their efficacy against a range of in vivo cancer models, including breast, pancreatic, and brain tumors.Jennifer C. Ashworth -holds an Anne McLaren fellowship at the University of Nottingham, UK.She was awarded her PhD in 2016 from the University of Cambridge, UK, where her research focussed on designing collagen scaffolds for regenerative medicine.Her current research centers on tissuemimetic biomaterials for 3D cell and organoid culture, with a particular focus on modeling the role of collagen organization in cancer and fibrosis.She is currently a Scientific Advisory Board member and co-founder of the University of Nottingham spin-out company PeptiMatrix.Anna M. Grabowska -was awarded her Ph.D. in 1989 by Cambridge University.She carried out postdoctoral research at Harvard Medical School, Cambridge University, and the University of Nottingham (UoN) and is now a Professor in Cancer Microenvironment, at UoN.She leads a group that has developed improved pre-clinical models of cancer that are more reflective of the tumor microenvironment and can be used to challenge new therapeutics in a more clinically relevant setting.These models incorporate early passage human cancer and stromal cells and make use of constitutive and inducible bioluminescent/fluorescent reporters to track cell proliferation and location and to relate the onset of biological processes to tumor progression and drug response.Cameron Alexander is Professor of Polymer Therapeutics at the School of Pharmacy, University of Nottingham, UK.Professor Alexander received degrees (B.Sc.and Ph.D.) in Chemistry from the University of Durham, UK, and carried out post-doctoral research at the Melville Laboratory for Polymer Synthesis, University of Cambridge.He is a Fellow of the Royal Society of Chemistry.His research focuses on polymer formulations for applications in areas ranging from vaccines and therapeutics for infectious diseases to cancers and neurodegeneration.Professor Alexander has been highly fortunate to work with scientists from more than 20 countries in his research group in the last decade.

Table 2 .
Preclinical studies for stimuli-responsive NPs in pancreatic cancer.