Cancer is a leading cause of morbidity and mortality worldwide, with recent advancements resulting in modest impacts on patient survival. Nanomedicine represents an innovative field with immense potential for improving cancer treatment, having ushered in several established drug delivery platforms. Nanoconstructs such as liposomes are widely used in clinics, while polymer micelles are in advanced phases of clinical trials in several countries. Currently, the field of nanomedicine is generating a new wave of nanoscale drug delivery strategies, embracing trends that involve the functionalization of these constructs with moieties that enhance site-specific delivery and tailored release. Herein, we discuss several advancements in established nanoparticle technologies such as liposomes, polymer micelles, and dendrimers regarding tumor targeting and controlled release strategies, which are being incorporated into their design with the hope of generating a more robust and efficacious nanotherapeutic modality. We also highlight a novel strategy known as multistage drug delivery; a rationally designed nanocarrier aimed at overcoming numerous biological barriers involved in drug delivery through the decoupling of various tasks that comprise the journey from the moment of systemic administration to arrival at the tumor site. (Cancer Sci 2011; 102: 1247–1252)
Cancer remains a cause of considerable morbidity and mortality worldwide, recently surpassing heart disease as the leading cause of death in the US population 85 years and younger.(1) More harrowing is the fact that several significant achievements towards the treatment of the disease have failed to profoundly impact patient survival. The last century witnessed the maturation of chemotherapy as a viable, adjuvant therapeutic modality for the treatment of cancer. With an enhanced understanding of the underlying mechanisms of tumorigenesis came the discovery and development of highly specific agents capable of exerting their effects on individual proteins or pathways either overexpressed or aberrant within tumors. This cell killing specificity was meant to sidestep toxicities associated with more traditional chemotherapeutics, while resulting in improved antitumor efficacies. As an example, human epidermal growth factor receptor 2 (HER2) was found to be overexpressed in one-fourth of breast tumors,(2) leading to the development of trastuzumab (Herceptin), a recombinant monoclonal antibody that binds to the extracellular domain of HER2.
While these novel chemotherapeutics have led to improvements in survival, they are still plagued by a number of biological barriers that hinder efficacious drug delivery following systemic administration. Novel and traditional chemotherapeutics suffer from non-specific distribution, with only a small fraction of drugs reaching the tumor. It is now well known that injected materials suffer from sequestration by the reticuloendothelial system (RES), a system comprised of monocytes and macrophages that clear foreign materials.(3) As a result, drugs accumulate in healthy organs, with their inherent toxicity drawing a fine line between tolerability and severe morbidity, as is the case of doxorubicin, a DNA intercalator that results in cardiotoxicity.(4) In addition to these, there is abnormal blood flow in and around tumors, interstitial pressure gradients, and cellular/nuclear membrane traversals to name a few. Taken together, these factors preclude the curative potential of anticancer drugs, warranting more effective ways to deliver them to tumors.
With these limitations in mind, and in attempts to apply Paul Ehrlich’s “magic bullet” concept to chemotherapy,(5) liposomes and polymer-drug conjugates were developed in the 1960s and 1970s.(6) These are now mainstay platforms within the field of nanomedicine, a multidisciplinary field that aims to utilize nanoscale (1–100 nm) constructs to improve delivery of chemotherapeutics.(7) Primordially, these carriers assist in drug solubilization and protect the drug from degradation. The nanoscale size range, and the oftentimes ubiquitous presence of poly(ethylene glycol) (PEG) on their surface, aids in evasion of the RES, allowing drugs to accumulate in tumors through the enhanced permeability and retention (EPR) effect, the result of tumor blood vessel leakiness.(8) Lower sequestration rates in healthy organs, coupled with higher accumulation and retention rates in tumors, translates directly to the prospect for higher efficacies and minimal patient side-effects.
The potential of nanomedicine for improving cancer care has begun to be realized clinically in several notable instances. In 2008, a team led by Davis(9) performed the first phase I clinical trial in human patients with solid cancers of a systemically administered targeted nanoparticle containing siRNA. Nanoparticles consisting of a linear, cyclodextrin-based polymer (CDP), transferrin (Tf) protein targeting ligands, and anti-R2 siRNA were shown to accumulate in tumor cells at therapeutically relevant doses. Most importantly, the platform resulted in mRNA cleavage at the site of RNA interference, demonstrating specific gene inhibition by systemically administered siRNA in humans. Herein, we aim to highlight similar innovative nanomedicine strategies, focusing on platforms and novel trends that are marshalling the next generation of chemotherapeutic formulations.
Established nanoparticle platforms
Liposomes. Liposomes are nanoconstructs (approximately 100 nm in diameter) with bilayered membrane structures composed of phospholipids with hydrophilic heads and hydrophobic anionic or cationic long-chain tails (Fig. 1A).(10) Their aqueous reservoir allows for encapsulation of a wide variety of hydrophilic agents, including drugs and siRNA.(11) Moreover, the hydrophobic membrane can encapsulate hydrophobic drug molecules and prevent leakage of hydrophilic agents from within the core. In 1995, the FDA approved the use of a PEGylated liposomal formulation of doxorubicin (Doxil) for the treatment of Kaposi’s sarcoma.(12,13) The platform resulted in significantly decreased cardiotoxicity and increased the circulation half-life of doxorubicin from 10 min to 50 h.(14) In a study by SEQUUS Pharmaceuticals, 53 patients with advanced Kaposi’s sarcoma were administered liposomal doxorubicin once every 3 weeks, with 19 patients showing a partial response and one experiencing a complete response.(15) The success of liposomal doxorubicin paved the way for several other liposomal formulations currently in various phases of clinical trials. LErafAON is a liposomal formulation containing the raf antisense oligonucleotide that inhibits c-raf, a protein that leads to resistance to radiation and chemotherapy.(16) In a phase I trial in patients with advanced solid tumors undergoing radiation therapy, four of 12 evaluable patients exhibited a partial response, while four showed stable disease. Three of five evaluable patients exhibited c-raf-1 mRNA inhibition and four of five exhibited Raf-1 protein inhibition.
Polymer micelles. Polymer micelles are spherical nanostructures, ranging from 10 to 100 nm, formed from the self-assembly of amphiphilic-block copolymers in aqueous environments (Fig. 1B).(17) As a result of this self-assembly, polymer micelles consist of a PEG hydrophilic corona and a hydrophobic core, typically composed of polymers like poly(ε-caprolactone) (PCL) and poly(d,l-lactic acid) (PLA), which allows for solubilization of lipophilic drugs.(18) Moreover, polymer micelles can be functionalized with targeting ligands for enhanced tumor accumulation strategies, or incorporate custom core-forming polymers for controlled degradation. Several micellar formulations are currently undergoing various phases of clinical trials. A cisplatin-incorporating polymeric micelle, NC-6004, was recently examined in a phase I clinical trial in patients with varying solid tumor types.(19) Of 17 patients treated, seven had stable disease, with micelles found to be well tolerated by patients. A paclitaxel-containing PEG-PLA micelle, Genexol-PM, was examined in a phase II clinical trial in patients with metastatic breast cancer.(20) Following administration of 300 mg/m2 every 3 weeks in 41 patients, the overall response rate was 58.5% with 19 partial responses and five complete responses.
Dendrimers. Dendrimers are nanoparticles (approximately 10 nm in size) composed of multiple units with functionalizable terminal groups branching out from a central core (Fig. 1C).(21) These nanoconstructs can incorporate multiple and varying therapeutic agents, either through conjugation to functional end groups,(22) or encapsulation within the central cavity and/or multiple channels between dendrons. Controlled depolymerization of the dendrimer results in highly tailorable release profiles. The chemical constitution of dendrimers, commonly composed of polyamidoamine (PAMAM), can be modified to increase biocompatibility or enhance tumor targeting through attachment of ligands. Dendrimers are showing immense potential for cancer therapy in preclinical studies. A recent study involving polylysine dendrimers aimed to target the activated α5β1 integrin, an integrin found to be crucial in tumor invasion and metastasis.(23) Yao and coworkers demonstrated that a dendrimer formulation containing the Pro-His-Ser-Cys-Asn (PHSCN) peptide, which binds to activated α5β1, hindered MDA-MB-231 tumor extravasation in the lungs of mice to a degree of 700- to 1100-fold more than the free peptide. Moreover, the PHSCN dendrimer reduced the number of established lung colonies following metastasis.
Novel prospects in nanomedicine for enhanced tumor treatment
Active targeting strategies. While “passive targeting” of nanoparticles results in increased preferential accumulation in tumors, significant non-specific uptake of circulating nanoparticles still occurs in healthy organs. Thus, an active area of research involves functionalization of nanoparticles with targeting moieties that identify and bind to receptors overexpressed on either tumors or their associated endothelium, consequently maximizing localization and accumulation in tumors (Fig. 2). Folate is a small organic molecule with a high affinity for the folic acid receptor, oftentimes overexpressed in certain tumors 100- to 300-fold over endogenous levels.(24) Singh and coworkers developed folate-functionalized PAMAM dendrimers containing 5-fluorouracil (5-FU) and examined its efficacy in mice bearing KB cells.(25) The PEGylated folate-functionalized dendrimers were shown to have high tumor accumulations of 20.1% and 10% of the injected dose at 8 and 24 h, respectively, resulting in a significant (approximately 40%) reduction in tumor growth compared with non-targeted controls after two injections (days 0 and 7) over 20 days.
While currently used in clinics as a therapeutic, trastuzumab can also function as a targeting moiety. Recently, liposomes encapsulating topotecan, a topoisomerase-I inhibitor, were functionalized with an anti-HER2 scFv F5 antibody for targeting BT-474 breast tumors.(26) Following a treatment regimen consisting of intravenous injections of a dose of 5 mg/kg of liposomes on days 14, 18 and 21 after tumor implantation, anti-HER2-targeted liposomes showed over a twofold enhancement in antitumor activity compared with non-targeted liposomes, and a greater than fivefold difference compared with free topotecan after 53 days.
Peptides consist of a sequence of several α-amino acids, with many target integrins, such as the transmembrane protein αvβ3, found overexpressed on blood vessels undergoing angiogenesis.(27) The cyclic(Arg-Gly-Asp-D-Phe-Lys) (cRGD) peptide binds, to a high degree, to the αvβ3 integrin, serving as a potential candidate for active targeting of nanoparticles. Recently, Nasongkla and coworkers demonstrated functionalization of doxorubicin-containing PEG-PLA polymer micelles with the cRGD targeting ligand.(28) Micelles showed enhanced cellular uptake in αvβ3-overexpressing SLK endothelial cells when compared with non-targeted micelles. In vivo examination in mice bearing A549 lung tumors showed that cRGD-labeled micelles containing superparamagnetic iron oxide nanoparticles effectively targeted and accumulated in tumor tissue to a greater extent than non-targeted controls, as evidenced by superior MRI imaging contrast.(29)
Controlled release strategies. Current research aims at tailoring release of drugs from nanoparticles in the hope of rapidly exposing the tumor to therapeutic levels of the drug, after which these levels are sustained. Due to their heavy dependence on glycolysis, tumors tend to have lower pH values than physiological levels, opening several avenues for controlling release. Talelli and coworkers developed a pH-sensitive polymer micelle with a covalently entrapped doxorubicin methacrylamide derivative within the core of poly(ethylene glycol)-b-poly[N-(2-hydroxypropyl) methacrylamide-lactate] (mPEG-b-p[HPMAm-Lacn]) micelles.(30) At a pH of 5.0, 100% of the drug was released after 24 h, whereas approximately 5% was released at pH 7.4 over the same time period. pH-sensitive micelles were more efficacious in B16F10 and OVCAR-3 ovarian cancer cells in vitro, which is likely to be the result of heightened release in acidic organelles. Moreover, in mice bearing B16F10 tumors, pH-sensitive micelles showed a greater than twofold difference in tumor growth suppression over PBS and free doxorubicin controls in a time span of 8 days after injections on days 3 and 6.
Ultrasound is gaining momentum as a strategy to induce site-specific release of therapeutic agents from nanoparticles (Fig. 2). The mechanism relies on the ability of ultrasound to produce free radicals that degrade polymers, increase local temperatures, and permeabilize cell membranes.(31) Schroeder and coworkers recently demonstrated ultrasound-triggered drug release of cisplatin from liposomes in vivo.(32) Liposomes were injected intraperitoneally into mice bearing J6456 lymphomas, followed by administration of low frequency ultrasound 1 h later. Results show that approximately 70% of cisplatin was released following ultrasound, compared with approximately 3% without ultrasound. In the tumors receiving ultrasound, cisplatin levels were nearly threefold higher than those in tumors receiving no ultrasound. In C-26 colon tumors in the footpad of mice, liposomes were injected intravenously, followed by ultrasound irradiation 24 h later. Findings demonstrate that tumor growth in the footpad was significantly suppressed in mice receiving cisplatin-containing liposomes and ultrasound when compared with free cisplatin with and without ultrasound after 29 days.
Externally induced temperature increases are a potential avenue to induce drug release in tumors. Incorporation of polymers, such as poly(N-isopropylacrylamide) (pNIPAM), possessing a lower critical solution temperature (LCST) leads to nanoparticle destabilization above this temperature due to hydrophobic effects.(33) Kono and coworkers developed thermosensitive doxorubicin liposomes incorporating poly(2-[2-ethoxy]ethoxyethyl vinyl ether [EOEOVE]), a copolymer with an LCST of 40°C.(34) Results demonstrate that at a temperature of 37°C, doxorubicin release from thermosensitive liposomes was minimal (<10% over 30 min). At 45°C, approximately 90% of the drug was released within the first 10 min. At different time points following intravenous injection of the thermosensitive liposomes in mice bearing subcutaneous C-26 colon cancer cells, the tumor was locally heated to 45°C for 10 min. Significant tumor reduction was observed in tumors subjected to local temperature increases at 3, 6 and 12 h following injection of liposomes compared with controls, with almost a 10-fold difference in tumor size observed 10 days following heating 12 h after injection.
Multistage drug delivery. Our laboratory has recently developed a novel multistage drug delivery strategy capable of not only circumnavigating several biological barriers encountered by nanoparticles en route to the tumor, but also of maximizing site-specific localization and release of therapeutics therein.(35,36) The rationale for this approach lies in the realization of mass transport differentials encountered in cancer, and the need to apply an understanding of “oncophysics” to overcome biobarriers.(37) The ultimate premise involves encapsulating drug-containing nanoconstructs within mesoporous silicon particles (MSP) that protect and ferry these nanoparticles until they recognize and dock at the tumor vasculature (Fig. 3). Hence, the platform partitions the individual tasks of drug delivery from the time of injection to arrival at the tumor site. The components of the platform are the following: (i) mesoporous silicon particles (also known as the first stage) that will be injected into the bloodstream and house; (ii) nanoparticles (also known as the second stage) loaded with; (iii) anticancer therapeutics (also known as the third stage). Biocompatible porous silicon was chosen as the housing material of the first stage because it is highly biodegradable under physiological conditions and has received FDA approval. Porous silicon nanostructures degrade into harmless silicic acid byproducts, presenting fewer challenges for long-term use. The size and shape of MSP are dictated by photolithographic masks, whereas the pore dimensions and porosity are adjusted through chemical composition and anodization conditions. Based on protocols developed in our laboratory, the outer dimension of the particles typically ranges from 500 nm to 1.6 μm in size with different geometries, and the mean pore size can range from 5 to 80 nm.(36,38) Their large size allows for encapsulation of a large payload of nanoparticles, while the versatility of the silicon chemistry allows for its functionalization with PEG and/or targeting moieties for increased tumor accumulation. No aggregation occurs following intravenous injection in mice, presenting a negligible risk for thrombosis. Moreover, the second stage can be any of a wide selection of nanoparticles, alone or in different combinations. To highlight this, two different particle types, quantum dots (QD) and PEG-FITC-single-walled carbon nanotubes (SWNT), were simultaneously loaded within MSP (Fig. 4A).(36) As observed, the SWNT (green) and QD (red) were found to co-localize within a single MSP, albeit, distributed in different regions; SWNT were found distributed throughout the MSP, while QD concentrated in the center of the particle.
Advantages afforded by the multistage approach for drug delivery are easily appreciable and lie in its multifunctionality and its ability to severely alter the pharmacokinetics of injectables. Using an integrated approach combining in silico mathematical modeling with in vitro and in vivo experimentation, MSP were rationally designed with specific geometries and sizes to maximize localization within tumor vasculature while minimizing RES uptake.(39) To accomplish this, the vascular journey of the MSP was compartmentalized, mathematically, into three main areas: (i) margination dynamics; (ii) firm adhesion; and (iii) control of internalization. In vitro experiments, in which particles of different shapes were injected in a parallel-plate flow chamber under controlled hydrodynamic conditions, showed that non-spherical particles exhibit a fairly complex behavior when in circulation, drifting laterally within the blood flow. This in turn increases the probability of interacting with blood vessel walls and facilitates binding of particles with specific vascular targets to a greater extent than for spherical particles. Further, oblate-spherical particles that resemble the shape of platelets tend to show more firm adherence to vessel walls under flow compared with spherical particles with the same number of ligand-receptor bonds. In vivo biodistribution studies demonstrated that particles of different shapes injected intravenously into mice bearing MDA-MB-231 breast tumors possess disparate in vivo fates (Fig. 4B).(40) Hemispherical particles showed the highest accumulation in tumors compared with spherical, discoidal and cylindrical particles. Taken together, these findings regarding hemodynamic properties and biodistribution significantly favor MSP accumulation in tumor-associated vessels, and emphasize the importance of taking into account specific geometries of nanocarriers for site-specific drug delivery applications.
Following particle migration to the tumor vasculature and microenvironment, cellular interactions ultimately dictate efficacy. The effect of particle surface charge, serum opsonization and inflammation on particle association with vascular endothelial cells was examined in vitro.(41) Vascular endothelial cells were shown to rapidly internalize, via both phagocytosis and macropinocytosis, both negatively (oxidized) and positively aminopropyltriethoxysilane (APTES)-modified charged MSP (Fig. 4C). Findings also demonstrate that following serum opsonization, APTES-modified MSP were more heavily internalized by endothelial cells. Another important property for MSP efficacy is adequate release of the therapeutic payload to achieve maximal cell-killing effects. Results show that release of second stage nanoparticles from the MSP is largely dependent on biodegradation of porous multistage particles under physiological conditions, resulting in the ability to tailor the release kinetics simply by fine-tuning the porosity of the MSP.(36) Highly porous multistage particles were shown to degrade within hours compared with particles with low porosity, which take days to degrade, enabling sustained, long-term release of the payload. Moreover, degradation was found to be dependent on surface functionalization of MSP.(42)
The efficacy of the multistage drug delivery approach was examined in a mouse model of ovarian cancer (SKOV3ip1), with the second stage comprising liposomes encapsulating siRNA against EphA2, an oncogenic tyrosine kinase receptor.(43) Liposomes were effectively loaded into MSP, providing a sustained release of siRNA (approximately 80% after 2 weeks). Mice were intravenously administered siRNA-liposomes biweekly for 3 weeks at a dose of 5 μg EphA2-siRNA per injection. A separate group was given a single administration of multistage EphA2-siRNA-liposomes at a dose of 15 μg EphA2-siRNA. The multistage delivery strategy was shown to induce sustained gene silencing of SKOV3ip1 tumor EphA2 protein for 3 weeks following a single administration, resulting in a heightened therapeutic response (Fig. 4D). As highlighted, treatment with EphA2-siRNA-liposomes in multistage significantly reduced tumor weight by 54.2% and 65.3% compared with non-silencing control siRNA-liposome and multistage-non-silencing control-siRNA-liposomes, respectively. It is important to note that the antitumor efficacy of a single administration was comparable to six repeated i.v. injections of EphA2-siRNA-liposomes. This result is likely the combination of improved biodistribution of the entrapped liposomal siRNA formulation, as well as the sustained release of these liposomes at the tumor site.
Recently, in the hope of achieving a theranostic nanoplatform, one that incorporates both therapeutic and imaging moieties within the same construct, MRI contrast agents were encapsulated within MSP and the magnetic properties examined.(44) Different formulations of the T1 contrast agent, gadolinium, including Magnevist (Bayer Schering Pharma, Berlin, Germany), gadofullerenes and gadonanotubes, were encapsulated in MSP. Once encapsulated, the gadolinium-loaded MSP demonstrated an enhanced longitudinal proton relaxivity (Fig. 4E), a direct correlate with improved image contrast. In some cases, these relaxivity values were 4–50 times greater than commercially available formulations, and were likely the direct result of nanoconfinement and clustering of the gadolinium within the MSP. These preliminary findings serve to highlight the immense potential that multistage delivery has in efficaciously treating and diagnosing cancer.
Chemotherapy is a powerful adjuvant therapy, but the ability to amass large amounts of drugs site-specifically remains elusive. While nanomedicine platforms such as liposomes and polymer micelles have improved delivery of anticancer agents, several biological barriers remain that limit antitumor efficacy. Current trends in nanomedicine involve incorporation of targeting moieties highly specific for receptors overexpressed in tumors (e.g. folic acid), and components that enable tailorable release at the site (e.g. ultrasound). Recently, we explored a multistage drug delivery approach, wherein a mesoporous silicon carrier particle is tasked with overcoming these biological barriers and transporting drug-containing nanoparticles to the tumor site. Increased targeting through size and geometry, enhanced protection of loaded cargo, and sustained release of therapeutics has directly translated to improved antitumor efficacy, highlighting the potential of this interdisciplinary approach for chemotherapy. The future of nanomedicine will no doubt yield innovative platforms for tumor treatment, and the giant strides presented herein will surely inspire these, potentially resulting in the personalization of chemotherapy for improved patient outcomes.
The authors are supported by DOD/BCRP-W81XWH-09-1-0212 to M.F.
M.F. serves on the Board of Directors of Arrowhead Research Corporation (NASDAQ:ARWR), Leonardo Biosystems, and NanoMedical Systems, and has a financial interest in these companies as a shareholder.