Nanoparticle drug delivery to target breast cancer brain metastasis: Current and future trends

Breast cancer brain metastasis (BCBM) is rapidly becoming an impediment to continuing survival gains seen in breast cancer patients. Drug delivery across the blood‐brain barrier is the main issue hindering systemic therapy against BCBM. This review details recent advances in nanoparticle (NP) drug delivery systems to target BCBM. Their primary benefits are: enhanced circulating and intra‐BCBM drug biodistribution, BCBM targeting through NP functionalization, opportunities for gene manipulation and their theragnostic applications. Multiple NPs have been synthesized to deliver therapeutic HER2 blockade, which is particularly important given HER2‐positive breast cancer's tendency to form BCBM. Finally, we review the clinical context in which NP‐based therapeutics have been investigated in BCBM patients. While a breakthrough in improving patient outcomes remain awaited, these clinical trials represent positive steps in the changing attitude towards BCBM as a treatable illness. Although multiple challenges remain in the clinical translation of BCBM‐directed NP therapies, ongoing research in the field offers promising avenues for novel targeting of this devastating disease.

growth factor receptor 2 (HER2) overexpressing (up to 28.7% of cases) subtypes as having a particular propensity to spread to the brain. 3,4 The incidence of BCBM is rising, largely due to increased use of neuroimaging associated with clinical trials and improved survival of patients with metastatic breast cancer. Therefore, developing effective treatment strategies against BCBM is imperative for maintaining survival and quality of life for these patients. 5 Brain localized therapies, in the form of whole brain radiotherapy (WBRT), stereotactic radiosurgery (SRS) and surgical resection remain the most effective treatments available for BCBM. Surgery is the most common form of treatment for solitary BCBM and is usually followed by a form of radiotherapy. 6 While both SRS and WBRT have a similar efficacy on overall survival, numerous studies have shown that WBRT is associated with more adverse effects such as cognitive deterioration compared to SRS. 7 Cytotoxic chemotherapy and molecularly targeted systemic agents are frequently used in conjunction with the brain localized therapies. However, the therapeutic efficacy of most drugs in the brain is limited by poor penetration through the blood-brain barrier (BBB), when administered through the systemic circulation. Moreover, the therapeutic benefit of many systemic agents against BCBM is generally less clear due to the historic practice of systematically excluding these patients from clinical trials.
In the context of HER2-amplified BCBM, trastuzumab is a highly effective treatment for patients with metastatic HER2-positive breast cancer, although patients receiving trastuzumab have been shown to have a higher incidence of BCBM. 7 Due to its nature as an anti-HER2 antibody with a high molecular weight (148 kDa), trastuzumab has low BBB penetration. 8 Lapatinib is a small molecule drug that is a dual tyrosine kinase inhibitor of epidermal growth factor receptor (EGFR) and HER2. Preclinical evidence of lapatinib biodistribution in experimental BCBM indicates that lapatinib poorly passes the BBB, with average concentrations in BCBM at most reaching 20% of concentrations in extracranial metastases. 9 In a clinical pharmacokinetic study, using drug concentration in cerebrospinal fluid (CSF) as a surrogate for BBB penetration, Gori et al found that orally administered lapatinib resulted in low CSF levels; although intracranial activity against BCBM and higher uptake of radiolabeled lapatinib were observed suggesting improved penetrance at the tumor-disrupted BBB. 10

| CHALLENGES IN THERAPEUTIC DRUG DESIGN FOR BCBM
The BBB is a highly selective, semipermeable boundary that physically and functionally separates the systemic and cerebral circulations. By restricting and preventing the free entry of water-soluble substances and large molecules from entering the brain, the BBB imposes unique challenges in effective delivery of drugs to the brain. A prime example of limited drug efficacy against BCBM is illustrated by trastuzumab, a monoclonal antibody that specifically binds to the HER2 protein expressed on the surface of breast cancer cells and inhibits cell proliferation. 6 Like most monoclonal antibodies, trastuzumab does not cross the BBB. 11 However, various studies have shown that when the BBB has been disrupted by either radiotherapy or surgery, the penetration of trastuzumab increases. [12][13][14] Several mechanisms, such as the presence of the tight junctions between endothelial cells and various transport channels that regulate the movement of substance across the BBB, 15 prevent the entry of many small molecule drugs as they have shown to become substrates of efflux transporters, resulting in limited penetration. 7 Due to the lack of transcellular or paracellular channels, the BBB permits three routes for molecules to gain access to the brain interstitial fluid, through (a) receptor-mediated transport through the BBB, (b) lipid-mediated free diffusion through the BBB or (c) via carrier-mediated transport systems. 16,17 P-glycoprotein (P-gp), expressed on the endothelial cell surface, is responsible for expelling toxins from the intracellular to the extracellular space via an adenosine triphosphate (ATP) activated process, further limiting the intratumoral drug concentration. 18 High expression of P-gp on tumor cells is also proposed to be one of the main causes of multiple drug resistance in cancer. 19 In primary and secondary brain tumors, the BBB is modified to form the blood-tumor barrier (BTB). 20 The BTB is characterized by a mixture of disorganized network of defective tumor-associated capillaries and original brain capillaries co-opted by tumor cells, with anatomical and physiological differences that are distinct from the BBB. [21][22][23] The BTB has been termed "leaky" in comparison to the BBB, as it allows the movement of large molecules such as antibodies; however, it is also characterized by significant intratumoral and intertumoral heterogeneity. 24 Hence, developing alternative approaches to treat BCBM, specifically to overcome the challenges of drug delivery, is a major source of research activity. One emerging treatment option is to use nanoparticles (NPs) to overcome some of the challenges posed by the BBB/BTB in delivering drugs directly to the site of the secondary brain tumor.

| ROLE OF NANOPARTICLES IN BREAST CANCER AND CNS DRUG THERAPY
Nanoparticles belong to a class of ultrafine materials, measuring between 1 and 100 nm in two or more dimensions. Due to their extremely small size, NPs exhibit unique physicochemical properties that markedly differ from equivalent larger scale materials. When used for drug delivery, the therapeutic payload can be dissolved, encapsulated, entrapped or conjugated to the nanoparticle. 25 Currently, NPs have been used to overcome a variety of pharmacokinetic shortcomings associated with systemic anticancer treatment, such as drug instability, side effects and nonspecific cell-targeting. 26 NPs have been shown to improve the pharmacokinetic profile of systemic therapies; for example, they are able to maintain an effective dose ratio of combined drugs and are capable of accumulating at the tumor site due to the enhanced permeability and retention (EPR) effect. 27 More recently, the use of functionalized nanoparticles to deliver drugs has become widely appreciated due to its ability to precisely home to target tissues and allow a controlled release of the therapeutic payload. 28 There are various materials from which nanoparticles can be created, such as lipids, polymer and viral particles. 29 Table 1 provides a brief overview about the various types of NPs that can be used as drug carrier systems; outlining the relative advantages and disadvantages of each NP system. NPs may be coated by a polymer which releases the drug from the core across the polymeric membrane via controlled diffusion or erosion. 27 The polymeric membrane can be made of a variety of material such as liposomes, that contain the drug within the membrane or drugs can be conjugated to gold particles via ionic or covalent bonding. 28 Viral particle-based NPs will not be discussed further in this review, as they have not yet been shown to be applicable in the management of BCBM.
The rising incidence of BCBM places greater urgency in the need to find new treatment strategies against this disease. With the wealth of therapeutic drugs that are known to be effective in metastatic breast cancer, overcoming the challenges of CNS delivery via the systemic circulation is an attractive means to rapidly widen our armamentarium against BCBM. In light of the opportunities provided by NPs in improving CNS drug delivery, in this review we will examine the studies investigating NPs as a drug delivery system in BCBM and discuss the ongoing challenges in the application of NPs in BCBM. Our aim was to describe the current landscape and to provide fresh impetus to ongoing research efforts in this area.

| METHODS
Using the following search terms "breast cancer," "brain metastasis" comprising a lipid domain stabilized with a copolymer, to deliver docetaxel. 30 These docetaxel-loaded nanoparticles showed rapid uptake by breast cancer cells, more prolonged drug circulation time and elevated brain bioavailability which significantly inhibited brain metastasis development and prolonged animal survival. Furthermore, the amphiphilic nanoparticles were shown to cross the BBB via a process of endogenous lipidation along with apolipoprotein E (ApoE). In another example with liposomal irinotecan, mice bearing BCBM showed prolonged plasma drug exposure and increased survival compared to mice treated with either liposomal vehicle alone or free irinotecan. 31 Figure 1 shows several mechanisms through which NPs can enhance drug biodistribution again BCBM.
The drug oxaliplatin is an anticancer drug that is rarely used to treat BCBM due to its lack of BBB penetrability. However, a preclinical study has shown that oxaliplatin encased by a liposomal NP resulted in increased plasma accumulation in mice bearing a subcutaneously engrafted human breast (MT-3) tumor compared to free drug.
Mice-bearing intracranial MT-3 tumors also had better tumor control when treated with liposomal oxaliplatin compared to either free drug or vehicle alone. 32 Similar findings, in intracranial MT-3 tumors, were found with mitoxantrone chemotherapy entrapped within fluid membrane liposomes that were functionalized to target the low-density lipoprotein receptor-related protein-1 (LRP1). 33 A study comparing polyethylene glycol (PEG) surface modified-or "PEGylated"-liposomal doxorubicin (PLD) with free doxorubicin, found that PLD had a 20-fold higher concentration in the intracranial tumor and 1500-fold higher plasma levels compared to the free form of the drug. PLD was still detectable in the circulation 96 hours later, but the free form was Using polymeric NPs containing camptothecin that were modified to target the transferrin receptor, Wyatt and Davis found that they were effective against different models of brain metastasis. 36 The authors also showed that the method for establishing BCBM, whether through intracranial, intracardiac or intravenous inoculation had a profound effect on the response to NP treatment, likely due to differences in the BBB/BTB. This finding raises an important implication for the design of preclinical studies, when testing NPs as a drug delivery system for BCBM.

| FUNCTIONALIZED NANOPARTICLES TO ENHANCE DRUG DELIVERY TO BCBM
Although the pharmacokinetic profile of NPs is favorable for increased drug delivery to the tumor, eliminating toxicity remains the holy grail for therapeutic drug design. One way to achieve this is by modifying the NPs to target the BCBM or its microenvironment. Khan  Ev E E asion from immune cell phagocyt y y osis F I G U R E 1 Different formulations of nanoparticles (NPs)-polymeric, liposomal and amphiphilic (inset image)-can enhance drug biodistribution against BCBM through several mechanisms. These mechanisms including improved penetration of the blood-brain barrier (BBB) due to the enhanced permeability and retention (EPR) effect and surface adsorption of circulating lipoproteins leading to receptor-mediated transcytosis (magnified image); reduction of drug efflux by active P-glycoprotein transport and avoidance of monocyte/macrophage destruction. Created with Biorender.com.
angiopep-2 (ANG), an oligopeptide that ligates LRP1, to augment LRP1 expression and, consequently, upregulate NP transcytosis through the BBB. 38 They showed that there was greater NP penetration with simvastatin pretreatment and significant improved survival in treated mice compared to free doxorubicin, functionalized NPs without doxorubicin or saline.
An alternative mechanism for enhancing NP transcytosis is through targeting the major facilitator superfamily domain-containing protein 2a (Mfsd2a), which is a key omega-3 fatty acid transporter that is exclusively expressed on brain endothelial cells. 39  A study generated polymer-lipid NPs conjugated to cyclic 9-amino acid internalizing peptide (iRGD) that were loaded with mitomycin C and doxorubicin. 47 Subsequently, the authors showed that treatment with these modified NPs not only reduced the brain metastatic burden of mice bearing triple-negative breast cancer (TNBC), but also had the dual benefit of reducing infiltration of tumor-associated macrophages, which are known to promote tumor growth. 48 Moreover, mice treated with the hybrid NP had more than a 50% increase in median survival compared to mice treated with the free form of the drugs. In a study con- inhibited the adhesion, migration and invasion of murine 4T1 mammary carcinoma cells more effectively compared to unbound lapatinib. 52 When tested in mice bearing intracarotid inoculated 4T1 brain metastases, besides improved brain penetration of LHNPs, the animals had more prolonged survival and reduced brain micrometastases when treated with LHNPs in a dose-dependent manner, compared to either orally or intravenously administered unbound lapatinib. F I G U R E 2 Functionalized NPs can enhance drug delivery to BCBM by several mechanisms: (1) paclitaxel-loaded polymer NPs conjugated with ITEM4 monoclonal antibody that targets fibroblast growth factor-inducible 14 (Fn14) on BCBM cells, while avoiding nonspecific binding to the brain extracellular matrix; (2) PLGA-PLL NPs co-functionalized with hyaluronic acid (HA) and transcytosis-targeting peptide (TTP) bind to heparin sulfate proteoglycans expressed on the blood-tumor barrier (BTB) and target the CD44-expressing BCBM cells, before releasing HAconjugated prodrug that is cleaved and activated by intracellular hyaluronidase; (3) mitomycin C/doxorubicin dual-loaded polymer-lipid hybrid NPs conjugated to cyclic 9-amino acid internalizing peptide (iRGD) that are transported across the BTB, through an integrin-mediated process, and inhibits tumor growth and reduces macrophage infiltration; (4) HA-targeted polymer NPs release minoxidil sulfate, which activates ATPsensitive potassium channels (K ATP ) expressed at the BTB and disrupts endothelial tight junctions, permitting BTB penetration by transcellular and paracellular routes; (5) doxorubicin-loaded PLGA NPs are encapsulated within a lipopolysaccharide-free bacterial outer membrane vesicle (dOMV) that permits BTB penetration and selective binding to tumor-expressed gp96; (6) doxorubicin/lapatinib dual loaded polymer NPs targeting endoluminal expressed prostate-specific membrane antigen (PSMA) with p32-assisted trafficking across the BTB; (7) tunicamycin-loaded NPs perform a priming strategy, by inhibiting BTB-expressed Mfsd2a function, allowing greater uptake of co-administered HA-targeted NPs to deliver doxorubicin; (8) fusion protein bound to drug-loaded liposomes that targets luminal low-density lipoprotein receptor-related protein-1 (LRP1) and is released in the abluminal side by matrix metalloproteinase-1 (MMP1) cleavage and (9)  ing results by using gene therapy via NPs. 62 In this case, polymer nanoparticles were used to inhibit microRNA10b (miRNA10b), which has been shown to play a role in tumor cell invasion, migration and metastatic initiation. The study found that mice injected with NP had a reduced brain metastatic burden compared to controls. Thus, NPs can be used as a delivery system for gene modification, opening up the therapeutic avenues against BCBM.

| Special applications of drug-loaded nanoparticles in breast cancer brain metastasis
Nanobioconjugation is a method that involves chemically bonding two molecules, with at least one being a biomolecule. An in vivo study These outcomes were backed by another clinical trial that found similar results. 68 The evaluation of NPs for management of BCBM, however, has been less mature; largely due to the challenges in recruiting these patients into clinical trials and the systematic exclusion of this patient group from clinical studies. Nevertheless, a case study has reported the long-term therapeutic potential of nab-paclitaxel in combination with trastuzumab for a patient with heavily pretreated metastatic HER2-positive BCBM, with associated disease stabilization of greater than 13 months. 69  with radiotherapy was safe and resulted in drug accumulation between 7 and 13 times higher in metastatic brain tumors than normal brain tissue. 73 Out of the 10 patients with metastatic brain tumors, three had primary breast cancer. Of these three breast cancer patients, two showed a complete response to the treatment, while the third had a partial response in the brain metastasis.
A phase II trial (NCT05255666) is currently in progress that will assess the combination treatment of liposomal irinotecan and Pembrolizumab for the treatment of triple-negative BCBM. This study shall hopefully provide valuable information on the use of NPs in the treatment of BCBM. The trial will measure primarily the disease control rate, along with secondary outcomes such as overall survival (OS) and PFS. More general problems associated with nanomedicines are the high cost and difficulty in manufacturing NPs that hinders scalable production necessary for clinical application. Technical challenges that need to be overcome by NPs include the risks of aggregation, fluid or protein adsorption, premature release of cargo, phase transition and contamination following contact with biological fluids. 78,79 Furthermore, the spontaneous coating of NPs by proteins, lipids and sugars, forming a protein corona, in the blood can inhibit the target specificity of functionalized NPs, as well as increasing susceptibility to degradation by the host innate immune system. 80,81 Therapeutic NPs have been shown to be successful drug delivery systems that can overcome the constraints on systemic drug penetration across the BBB. Mostly these have been tested on standard cytotoxic chemotherapy agents. Given the propensity of HER2-positive breast cancer to spread to the brain, the ability of NPs to deliver anti-HER2 targeted therapies is a potential gamechanger in this disease.
Beyond the role of NPs as vehicles for therapeutics, they have also been shown to be versatile tools for gene delivery and for theragnostic purposes against BCBM. negative subtype. 82,83 As an example, bioengineered NPs have been used to target bone marrow-derived immune cells and to train them to target cancer. 84 Thus, additional therapeutic options may be opened up for patients with TNBC and brain metastasis. NPs have also been proposed to be used as artificial antigen-presenting cells (aAPCs). Carbon nanotubes have previously been engineered as aAPCs for optimal interactions with T cells in mice. 85 Moreover, NPs may be combined with extracorporeal technology to enhance drug delivery at the target site, for example using focused ultrasound to disrupt the BTB and enhance the delivery of liposomal NPs. A study conducted by Wu et al, showed that the use of pulsed-wave and low-dose ultrasound significantly enhanced the delivery and antitumoral effects of NPs in BCBM bearing mice compared to controls. 86 With the projected rise in BCBM incidence, it is imperative that we begin to exploit the opportunity offered by NPs in enhancing drug delivery to the brain. The multitude of preclinical evidence for benefit of NPs in BCBM provides hope that additional systemic therapy options will soon become available for these patients.