EGFR Targeting of Liposomal Doxorubicin Improves Recognition and Suppression of Non-Small Cell Lung Cancer.

Introduction: Despite improvements in chemotherapy and molecularly targeted therapies, the life expectancy of patients with advanced non-small cell lung cancer (NSCLC) remains less than 1 year. There is thus a major global need to advance new treatment strategies that are more effective for NSCLC. Drug delivery using liposomal particles has shown success at improving the biodistribution and bioavailability of chemotherapy. Nevertheless, liposomal drugs lack selectivity for the cancer cells and have a limited ability to penetrate the tumor site, which severely limits their therapeutic potential. Epidermal growth factor receptor (EGFR) is overexpressed in NSCLC tumors in about 80% of patients, thus representing a promising NSCLC-specific target for redirecting liposome-embedded chemotherapy to the tumor site. Methods: Herein, we investigated the targeting of PEGylated liposomal doxorubicin (Caelyx), a powerful off-the-shelf antitumoral liposomal drug, to EGFR as a therapeutic strategy to improve the specific delivery and intratumoral accumulation of chemotherapy in NSCLC. EGFR-targeting of Caelyx was enabled through its complexing with a polyethylene glycol (PEG)/EGFR bispecific antibody fragment. Tumor targeting and therapeutic potency of our treatment approach were investigated in vitro using a panel of NSCLC cell lines and 3D tumoroid models, and in vivo in a cell line-derived tumor xenograft model. Results: Combining Caelyx with our bispecific antibody generated uniform EGFR-targeted particles with improved binding and cytotoxic efficacy toward NSCLC cells. Effects were exclusive to cancer cells expressing EGFR, and increments in efficacy positively correlated with EGFR density on the cancer cell surface. The approach demonstrated increased penetration within 3D spheroids and was effective at targeting and suppressing the growth of NSCLC tumors in vivo while reducing drug delivery to the heart. Conclusion: EGFR targeting represents a successful approach to enhance the selectivity and therapeutic potency of liposomal chemotherapy toward NSCLC.

Targeted delivery of polo-like kinase 1 siRNA nanoparticles using an EGFR-PEG bispecific antibody inhibits proliferation of high-risk neuroblastoma.

High-risk neuroblastoma has poor survival due to treatment failure and off-target side effects of therapy. Small molecule inhibitors have shown therapeutic efficacy at targeting oncogenic cell cycle dysregulators, such as polo-like kinase 1 (PLK1). However, their clinical success is limited by a lack of efficacy and specificity, causing off-target toxicity. Herein, we investigate a new treatment strategy whereby a bispecific antibody (BsAb) with dual recognition of methoxy polyethylene glycol (PEG) and a neuroblastoma cell-surface receptor, epidermal growth factor receptor (EGFR), is combined with a PEGylated small interfering RNA (siRNA) lipid nanoparticle, forming BsAb-nanoparticle RNA-interference complexes for targeted PLK1 inhibition against high-risk neuroblastoma. Therapeutic efficacy of this strategy was explored in neuroblastoma cell lines and a tumor xenograft model. Using ionizable lipid-based nanoparticles as a low-toxicity and clinically safe approach for siRNA delivery, we identified that their complexing with EGFR-PEG BsAb resulted in increases in cell targeting (1.2 to >4.5-fold) and PLK1 gene silencing (>2-fold) against EGFR+ high-risk neuroblastoma cells, and enhancements correlated with EGFR expression on the cells (r > 0.94). Through formulating nanoparticles with PEG-lipids ranging in diffusivity, we further identified a highly diffusible PEG-lipid which provided the most pronounced neuroblastoma cell binding, PLK1 silencing, and significantly reduced cancer growth in vitro in high-risk neuroblastoma cell cultures and in vivo in a tumor-xenograft mouse model of the disease. Together, this work provides an insight on the role of PEG-lipid diffusivity and EGFR targeting as potentially relevant variables influencing the therapeutic efficacy of siRNA nanoparticles in high-risk neuroblastoma.

An immunomodulating peptide with potential to suppress tumour growth and autoimmunity.

Cancers and autoimmune diseases commonly co-exist and immune checkpoint inhibitor therapy (ICI) exacerbates autoimmune pathologies. We recently described a lipidic peptide, designated IK14004, that promotes expansion of immunosuppressive T regulatory (Treg) cells and uncouples interleukin-2 from interferon-gamma production while activating CD8+ T cells. Herein, we report IK14004-mediated inhibition of Lewis lung cancer (LLC) growth and re-invigoration of splenocyte-derived exhausted CD4+ T cells. In human immune cells from healthy donors, IK14004 modulates expression of the T cell receptor α/ß subunits, induces Type I IFN expression, stimulates natural killer (NK) cells to express NKG2D/NKp44 receptors and enhances K562 cytotoxicity. In both T and NK cells, IK14004 alters the IL-12 receptor ß1/ß2 chain ratio to favour IL-12p70 binding. Taken together, this novel peptide offers an opportunity to gain further insight into the complexity of ICI immunotherapy so that autoimmune responses may be minimised without promoting tumour evasion from the immune system.

Targeted Hyperbranched Nanoparticles for Delivery of Doxorubicin in Breast Cancer Brain Metastasis.

Breast cancer brain metastases (BM) are associated with a dismal prognosis and very limited treatment options. Standard chemotherapy is challenging in BM patients because the high dosage required for an effective outcome causes unacceptable systemic toxicities, a consequence of poor brain penetration, and a short physiological half-life. Nanomedicines have the potential to circumvent off-target toxicities and factors limiting the efficacy of conventional chemotherapy. The HER3 receptor is commonly expressed in breast cancer BM. Here, we investigate the use of hyperbranched polymers (HBP) functionalized with a HER3 bispecific-antibody fragment for cancer cell-specific targeting and pH-responsive release of doxorubicin (DOX) to selectively deliver and treat BM. We demonstrated that DOX-release from the HBP carrier was controlled, gradual, and greater in endosomal acidic conditions (pH 5.5) relative to physiologic pH (pH 7.4). We showed that the HER3-targeted HBP with DOX payload was HER3-specific and induced cytotoxicity in BT474 breast cancer cells (IC50: 17.6 µg/mL). Therapeutic testing in a BM mouse model showed that HER3-targeted HBP with DOX payload impacted tumor proliferation, reduced tumor size, and prolonged overall survival. HER3-targeted HBP level detected in ex vivo brain samples was 14-fold more than untargeted-HBP. The HBP treatments were well tolerated, with less cardiac and oocyte toxicity compared to free DOX. Taken together, our HER3-targeted HBP nanomedicine has the potential to deliver chemotherapy to BM while reducing chemotherapy-associated toxicities.

Exploring the impact of severity in hepatic fibrosis disease on the intrahepatic distribution of novel biodegradable nanoparticles targeted towards different disease biomarkers.

Nanoparticle-based drug delivery systems (DDS) have shown promising results in reversing hepatic fibrosis, a common pathological basis of chronic liver diseases (CLDs), in preclinical animal models. However, none of these nanoparticle formulations has transitioned to clinical usage and there are currently no FDA-approved drugs available for liver fibrosis. This highlights the need for a better understanding of the challenges faced by nanoparticles in this complex disease setting. Here, we have systematically studied the impact of targeting strategy, the degree of macrophage infiltration during fibrosis, and the severity of fibrosis, on the liver uptake and intrahepatic distribution of nanocarriers. When tested in mice with advanced liver fibrosis, we demonstrated that the targeting ligand density plays a significant role in determining the uptake and retention of the nanoparticles in the fibrotic liver whilst the type of targeting ligand modulates the trafficking of these nanoparticles into the cell population of interest - activated hepatic stellate cells (aHSCs). Engineering the targeting strategy indeed reduced the uptake of nanoparticles in typical mononuclear phagocyte (MPS) cell populations, but not the infiltrated macrophages. Meanwhile, additional functionalization may be required to enhance the efficacy of DDS in end-stage fibrosis/cirrhosis compared to early stages.

Delivery of PEGylated liposomal doxorubicin by bispecific antibodies improves treatment in models of high-risk childhood leukemia.

High-risk childhood leukemia has a poor prognosis because of treatment failure and toxic side effects of therapy. Drug encapsulation into liposomal nanocarriers has shown clinical success at improving biodistribution and tolerability of chemotherapy. However, enhancements in drug efficacy have been limited because of a lack of selectivity of the liposomal formulations for the cancer cells. Here, we report on the generation of bispecific antibodies (BsAbs) with dual binding to a leukemic cell receptor, such as CD19, CD20, CD22, or CD38, and methoxy polyethylene glycol (PEG) for the targeted delivery of PEGylated liposomal drugs to leukemia cells. This liposome targeting system follows a "mix-and-match" principle where BsAbs were selected on the specific receptors expressed on leukemia cells. BsAbs improved the targeting and cytotoxic activity of a clinically approved and low-toxic PEGylated liposomal formulation of doxorubicin (Caelyx) toward leukemia cell lines and patient-derived samples that are immunophenotypically heterogeneous and representative of high-risk subtypes of childhood leukemia. BsAb-assisted improvements in leukemia cell targeting and cytotoxic potency of Caelyx correlated with receptor expression and were minimally detrimental in vitro and in vivo toward expansion and functionality of normal peripheral blood mononuclear cells and hematopoietic progenitors. Targeted delivery of Caelyx using BsAbs further enhanced leukemia suppression while reducing drug accumulation in the heart and kidneys and extended overall survival in patient-derived xenograft models of high-risk childhood leukemia. Our methodology using BsAbs therefore represents an attractive targeting platform to potentiate the therapeutic efficacy and safety of liposomal drugs for improved treatment of high-risk leukemia.

Combination of Chemotherapy and Mild Hyperthermia Using Targeted Nanoparticles: A Potential Treatment Modality for Breast Cancer.

Despite the clinical benefits that chemotherapeutics has had on the treatment of breast cancer, drug resistance remains one of the main obstacles to curative cancer therapy. Nanomedicines allow therapeutics to be more targeted and effective, resulting in enhanced treatment success, reduced side effects, and the possibility of minimising drug resistance by the co-delivery of therapeutic agents. Porous silicon nanoparticles (pSiNPs) have been established as efficient vectors for drug delivery. Their high surface area makes them an ideal carrier for the administration of multiple therapeutics, providing the means to apply multiple attacks to the tumour. Moreover, immobilising targeting ligands on the pSiNP surface helps direct them selectively to cancer cells, thereby reducing harm to normal tissues. Here, we engineered breast cancer-targeted pSiNPs co-loaded with an anticancer drug and gold nanoclusters (AuNCs). AuNCs have the capacity to induce hyperthermia when exposed to a radiofrequency field. Using monolayer and 3D cell cultures, we demonstrate that the cell-killing efficacy of combined hyperthermia and chemotherapy via targeted pSiNPs is 1.5-fold higher than applying monotherapy and 3.5-fold higher compared to using a nontargeted system with combined therapeutics. The results not only demonstrate targeted pSiNPs as a successful nanocarrier for combination therapy but also confirm it as a versatile platform with the potential to be used for personalised medicine.

Development and Validation of a Targeted Treatment for Brain Tumors Using a Multi-Drug Loaded, Relapse-Resistant Polymeric Theranostic.

This study aimed to develop a multifunctional polymer platform that could address the issue of treatment resistance when using conventional chemotherapeutics to treat glioblastoma (GBM). An antibody-conjugated, multi-drug loaded hyperbranched polymer was developed that provided a platform to evaluate the role of targeted nanomedicine treatments in overcoming resistant GBM by addressing the various complications with current clinically administered formulations. The polymer was synthesized via reversible addition fragmentation chain transfer polymerization and included the clinical first-line alkylating agent temozolomide (TMZ) which was incorporated as a polymerizable monomer, poly (ethylene glycol) (PEG) units to impart biocompatibility and enable conjugation with αPEG-αEphA2 bispecific antibody (αEphA2 BsAb) for tumor targeting, and hydrazide moieties for attachment of a secondary drug which allows exploration of synergistic therapies. To overcome the resistance to TMZ, the O6 alkylguanine DNA alkyltransferase (AGT, DNA repair protein) inhibitor, dialdehyde O6 benzylguanine (DABG) was subsequently conjugated to the polymer via an acid labile hydrazone linker to facilitate controlled release under conditions encountered within the tumor microenvironment. The prolonged degradation half-life (4-5 h) of the polymer conjugated TMZ in vitro offered a potential avenue to overcome the inability to deliver these drugs in combination at therapeutic doses. Although only 20% of DABG could be released within the studied timeframe (192 h) under conditions mimicking the acidic nature of the tumor environment, cytotoxicity evaluation using cell assays confirmed the improved therapeutic efficacy toward resistant GBM cells after attaching DABG to the polymer delivery vehicle. Of note, when the polymeric delivery vehicle was specifically targeted to receptors (Ephrin A2) on the surface of the GBM cells using our in-house developed EphA2 specific BsAb, the dual-drug-loaded polymer exhibited an improved therapeutic effect on TMZ-resistant cells compared to the free drug combination. Both in vitro and in vivo targeting studies showed high uptake of the construct to GBM tumors with an upregulated EphA2 receptor (T98G and U251) compared to a tumor that had low expression (U87MG), where a dual tumor xenograft model was used to demonstrate the enhanced accumulation in tumor tissue in vivo. Despite the synthetic challenges of developing systems to effectively deliver controlled doses of TMZ and DABG, these studies highlight the potential benefit of this formulation for delivering multi-drug combinations to resistant GBM tumor cells and offer a platform for future optimization in therapeutic studies.

RAFT Polymer-Antibody Conjugation: Squaramide Ester Chemistry Leads to Conjugates with a Therapeutic Anti-EGFR Antibody with Full Retention of Activity and Increased Tumor Uptake In Vivo.

Covalent conjugation of a biologically stable polymer to a therapeutic protein, e.g., an antibody, holds many benefits such as prolonged plasma exposure of the protein and improved tumor uptake. Generation of defined conjugates is advantageous in many applications, and a range of site-selective conjugation methods have been reported. Many current coupling methods lead to dispersity in coupling efficiencies with subsequent conjugates of less-well-defined structure, which impacts reproducibility of manufacture and ultimately may impact successful translation to treat or image diseases. We explored designing stable, reactive groups for polymer conjugation reactions that would lead to conjugates through the simplest and most abundant residue on most proteins, the lysine residue, yielding conjugates in high purity and demonstrating retention of mAb efficacy through surface plasmon resonance (SPR), cell targeting, and in vivo tumor targeting. We utilized squaric acid diesters as coupling agents for selective amidation of lysine residues and were able to selectively conjugate one, or two, high-molecular-weight polymers to a therapeutically relevant antibody, 528mAb, that subsequently retained full binding specificity. Water-soluble copolymers of N-(2-hydroxypropyl) methacrylamide (HPMA) and N-isopropylacrylamide (NIPAM) were prepared by Reversible Addition-Fragmentation chain-Transfer (RAFT) polymerization and we demonstrated that a dual-dye-labeled antibody-RAFT conjugate (528mAb-RAFT) exhibited effective tumor targeting in model breast cancer xenografts in mice. The combination of the precise and selective squaric acid ester conjugation method, with the use of RAFT polymers, leads to a promising strategic partnership for improved therapeutic protein-polymer conjugates having a very-well-defined structure.

Self-cyclisation as a general and efficient platform for peptide and protein macrocyclisation.

Macrocyclisation of proteins and peptides results in a remarkable increase in structural stability, making cyclic peptides and proteins of great interest in drug discovery-either directly as drug leads or as in the case of cyclised nanodiscs (cNDs), as tools for studies of trans-membrane receptors and membrane-active peptides. Various biological methods have been developed that are capable of yielding head-to-tail macrocyclised products. Recent advances in enzyme-catalysed macrocyclisation include discovery of new enzymes or design of new engineered enzymes. Here, we describe the engineering of a self-cyclising "autocyclase" protein, capable of performing a controllable unimolecular reaction for generation of cyclic biomolecules in high yield. We characterise the self-cyclisation reaction mechanism, and demonstrate how the unimolecular reaction path provides alternative avenues for addressing existing challenges in enzymatic cyclisation. We use the method to produce several notable cyclic peptides and proteins, demonstrating how autocyclases offer a simple, alternative way to access a vast diversity of macrocyclic biomolecules.

Efficient delivery of Temozolomide using ultrasmall large-pore silica nanoparticles for glioblastoma.

The prognosis of brain cancers such as glioblastoma remains poor despite numerous advancements in the field of neuro-oncology. The presence of the blood brain barrier (BBB) along with the highly invasive and aggressive nature of glioblastoma presents a difficult challenge for developing effective therapies. Temozolomide (TMZ) is a first line agent used in the clinic for glioblastoma and it has been useful in increasing patient survival rates. However, TMZ suffers from issues related to its pharmacokinetics, such as a short plasma half-life (2 h), is subjected to P-gp efflux, and has limited extravasation from blood to brain (∼20%). It has been postulated that reducing its efflux and increasing glioblastoma tissue exposure to TMZ could prove useful in treating glioblastoma and preventing tumour recurrence. Herein, ultra-small, large pore silica nanoparticles (USLP) have been loaded with TMZ, surface PEGlyated to reduce efflux and decorated with the cascade targeting protein lactoferrin for efficient uptake across the BBB and into glioblastoma. Our results demonstrate that USLP improves permeability of BBB in vitro as evidenced using a transwell model which mimics endothelial tight junctions with permeation being enhanced using PEGylated particles. Data from TMZ loaded USLP in vitro transwell BBB model also suggests that the USLP formulations can significantly reduce the efflux ratio of TMZ. In vitro apoptosis studies on glioblastoma cell lines U87 and GL261 were conducted which showed an improvement in TMZ induced glioblastoma apoptosis with USLP formulations compared to pure TMZ. Finally, a proof-of-concept preclinical mouse study demonstrated that when given intravenously at 50 mg/kg, USLP particles showed accumulation in the brain within a few hours without any obvious pathophysiological changes in vital organs as assessed via histology. Overall, the data suggests our innovative delivery system is efficient in extravasation from blood and permeating the BBB and has potential to improve efficacy of TMZ in glioblastoma therapy.

Pharmacokinetics and Biodistribution of 89Zr-Miltuximab and Its Antibody Fragments as Glypican-1 Targeting Immuno-PET Agents in Glioblastoma.

Glioblastoma (GBM) is the most aggressive form of primary brain cancer, accounting for about 85% of all primary central nervous system (CNS) tumors. With standard treatment strategies like surgery, radiation, and chemotherapy, the median survival time of patients with GBM is only 12-15 months from diagnosis. The poor prognosis of GBM is due to a very high tumor recurrence rate following initial treatment, indicating a dire need for improved diagnostic and therapeutic alternatives for this disease. Antibody-based immunotheranostics holds great promise in treating GBM, combining the theranostic applications of radioisotopes and target-specificity of antibodies. In this study, we developed and validated antibody-based positron emission tomography (PET) tracers targeting the heparan sulfate proteoglycan, glypican-1 (GPC-1), for noninvasive detection of disease using diagnostic molecular imaging. GPC-1 is overexpressed in multiple solid tumor types, including GBM, and is a promising biomarker for novel immunotheranostics. Here, we investigate zirconium-89 (89Zr)-conjugated Miltuximab (a clinical stage anti-GPC-1 monoclonal antibody developed by GlyTherix, Ltd.) and engineered fragments for their potential as immuno-PET tracers to detect GPC-1positive GBM tumors in preclinical models. We explore the effects of molecular size, avidity, and Fc-domain on the pharmacokinetics and biodistribution in vivo, by comparing in parallel the full-length antibody (Miltuximab), Fab'2, Fab, and single-chain variable fragment (scFv) formats. High radiolabeling efficiency (>95%) was demonstrated by all the formats and the stability post-radiolabeling was higher for larger constructs of Miltuximab and the Fab. Receptor-mediated internalization of all 89Zr-labeled formats was observed in a human GBM cell line in vitro, while full-length Miltuximab demonstrated the highest tumor retention (5.7 ± 0.94% ID/g, day-9 postinjection (p.i.)) and overall better tumor-to-background ratios than the smaller Fc-less formats. Results from in vivo PET image quantification and ex vivo scintillation counting were highly correlated. Altogether, 89Zr-DFO-Miltuximab appears to be an effective immuno-PET imaging agent for detecting GPC-1positive tumors such as GBM and the current results support utility of the Fc containing whole mAb format over smaller antibody fragments for this target.

Utilizing PET and MALDI Imaging for Discovery of a Targeted Probe for Brain Endocannabinoid α/ß-Hydrolase Domain 6 (ABHD6).

Multimodal imaging provides rich biological information, which can be exploited to study drug activity, disease associated phenotypes, and pharmacological responses. Here we show discovery and validation of a new probe targeting the endocannabinoid α/ß-hydrolase domain 6 (ABHD6) enzyme by utilizing positron emission tomography (PET) and matrix-assisted laser desorption/ionization (MALDI) imaging. [18F]JZP-MA-11 as the first PET ligand for in vivo imaging of the ABHD6 is reported and specific uptake in ABHD6-rich peripheral tissues and major brain regions was demonstrated using PET. A proof-of-concept study in nonhuman primate confirmed brain uptake. In vivo pharmacological response upon ABHD6 inhibition was observed by MALDI imaging. These synergistic imaging efforts used to identify biological information cannot be obtained by a single imaging modality and hold promise for improving the understanding of ABHD6-mediated endocannabinoid metabolism in peripheral and central nervous system disorders.

A Homochiral Poly(2-oxazoline)-based Membrane for Efficient Enantioselective Separation.

Chiral separation membranes have shown great potential for the efficient separation of racemic mixtures into enantiopure components for many applications, such as in the food and pharmaceutical industries; however, scalable fabrication of membranes with both high enantioselectivity and flux remains a challenge. Herein, enantiopure S-poly(2,4-dimethyl-2-oxazoline) (S-PdMeOx) macromonomers were synthesized and used to prepare a new type of enantioselective membrane consisting of a chiral S-PdMeOx network scaffolded by graphene oxide (GO) nanosheets. The S-PdMeOx-based membrane showed a near-quantitative enantiomeric excess (ee) (98.3±1.7 %) of S-(-)-limonene over R-(+)-limonene and a flux of 0.32 mmol m-2 h-1 . This work demonstrates the potential of homochiral poly(2,4-disubstituted-2-oxazoline)s in chiral discrimination and provides a new route to the development of highly efficient enantioselective membranes using synthetic homochiral polymer networks.

Probing the Biocompatibility and Immune Cell Association of Chiral, Water-Soluble, Bottlebrush Poly(2-oxazoline)s.

Poly(2-oxazoline)s (POx) have received substantial attention as poly(ethylene glycol) (PEG) alternatives in the biomedical field due to their biocompatibility, high functionality, and ease of synthesis. While POx have demonstrated strong potential as biomaterial constituents, the larger family of poly(cyclic imino ether)s (PCIE) to which POx belongs remains widely underexplored. One highly interesting sub-class of PCIE is poly(2,4-disubstituted-2-oxazoline)s (PdOx), which bear an additional substituent on the backbone of the polymers' repeating units. This allows fine-tuning of the hydrophilic/hydrophobic balance and renders the PdOx chiral when enantiopure 2-oxazoline monomers are used. Herein, we synthesize new water-soluble (R-/S-/RS-) poly(oligo(2-ethyl-4-methyl-2-oxazoline) methacrylate) (P(OEtMeOxMA)) bottlebrushes and compare them to well-established PEtOx- and PEG-based bottlebrush controls in terms of their physical properties, hydrophilicity, and biological behavior. We reveal that the P(OEtMeOxMA) bottlebrushes show a lower critical solution temperature behavior at a physiologically relevant temperature (∼44 °C) and that the enantiopure (R-/S-) variants display a chiral secondary structure. Importantly, we demonstrate the biocompatibility of the chiral P(OEtMeOxMA) bottlebrushes through cellular association and mouse biodistribution studies and show that these systems display higher immune cell association and organ accumulation than the two control polymers. These novel materials possess properties that hold promise for applications in the field of nanomedicine and may be beneficial carriers for therapeutics that require enhanced cellular association and immune cell interaction.

Enhanced drug delivery to cancer cells through a pH-sensitive polycarbonate platform.

Polymer-drug conjugates are widely investigated to enhance the selectivity of therapeutic drugs to cancer cells, as well as increase circulation lifetime and solubility of poorly soluble drugs. In order to direct these structures selectively to cancer cells, targeting agents are often conjugated to the nanoparticle surface as a strategy to limit drug accumulation in non-cancerous cells and therefore reduce systemic toxicity. Here, we report a simple procedure to generate biodegradable polycarbonate graft copolymer nanoparticles that allows for highly efficient conjugation and intracellular release of S-(+)-camptothecin, a topoisomerase I inhibitor widely used in cancer therapy. The drug-polymer conjugate showed strong efficacy in inhibiting cell proliferation across a range of cancer cell lines over non-cancerous phenotypes, as a consequence of the increased intracellular accumulation and subsequent drug release specifically in cancer cells. The enhanced drug delivery towards cancer cells in vitro demonstrates the potential of this platform for selective treatments without the addition of targeting ligands.

Modulating Macrophage Clearance of Nanoparticles: Comparison of Small-Molecule and Biologic Drugs as Pharmacokinetic Modifiers of Soft Nanomaterials.

Nanomedicines show benefits in overcoming the limitations of conventional drug delivery systems by reducing side effects, toxicity, and exhibiting enhanced pharmacokinetic (PK) profiles to improve the therapeutic window of small-molecule drugs. However, upon administration, many nanoparticles (NPs) prompt induction of host innate immune responses, which in combination with other clearance pathways such as renal and hepatic, eliminate up to 99% of the administered dose. Here, we explore a drug predosing strategy to transiently suppress the mononuclear phagocyte system (MPS), subsequently improving the PK profile and biological behaviors exhibited by a model NP system [hyperbranched polymers (HBPs)] in an immunocompetent mouse model. In vitro assays allowed the identification of five drug candidates that attenuated cellular association. Predosing of lead compounds chloroquine (CQ) and zoledronic acid (ZA) further showed increased HBP retention within the circulatory system of mice, as shown by both fluorescence imaging and positron emission tomography-computed tomography. Flow cytometric evaluation of spleen and liver tissue cells following intravenous administration further demonstrated that CQ and ZA significantly reduced HBP association with myeloid cells by 23 and 16%, respectively. The results of this study support the use of CQ to pharmacologically suppress the MPS to improve NP PKs.

Investigation of a Dual siRNA/Chemotherapy Delivery System for Breast Cancer Therapy.

Multidrug resistance (MDR) is a problem that is often associated with a poor clinical outcome in chemotherapeutic cancer treatment. MDR may potentially be overcome by utilizing synergistic approaches, such as combining siRNA gene therapy and chemotherapy to target different mechanisms of apoptosis. In this study, a strategy is presented for developing multicomponent nanomedicines using orthogonal and compatible chemistries that lead to effective nanotherapeutics. Hyperbranched polymers were used as drug carriers that contained doxorubicin (DOX), attached via a pH-sensitive hydrazone linkage, and ataxia-telangiectasia mutated (ATM) siRNA, attached via a redox-sensitive disulfide group. This nanomedicine also contained cyanine 5 (Cy5) as a diagnostic tracer as well as in-house developed bispecific antibodies that allowed targeting of the epidermal growth factor receptor (EGFR) present on tumor tissue. Highly efficient coupling of siRNA was achieved with 80% of thiol end-groups on the hyperbranched polymer coupling with siRNA. This attachment was reversible, with the majority of siRNA released in vitro under reducing conditions as desired. In cellular studies, the nanomedicine exhibited increased DNA damage and cancer cell inhibition compared to the individual treatments. Moreover, the nanomedicine has great potential to suppress the metabolism of cancer cells including both mitochondrial respiration and glycolytic activity, with enhanced efficacy observed when targeted to the cell surface protein EGFR. Our findings indicated that co-delivery of ATM siRNA and DOX serves as a more efficient therapeutic avenue in cancer treatment than delivery of the single species and offers a potential route for synergistically enhanced gene therapy.

Confined microemulsion sono-polymerization of poly(ethylene glycol) nanoparticles for targeted delivery.

Confined sono-polymerization is developed to prepare poly(ethylene glycol) nanoparticles within water-in-oil microemulsion, followed by post-functionalization with a bispecific antibody (anti HER2 and anti PEG) for targeted delivery of photosensitizers (i.e., indocyanine green). The nanoparticles could specifically target to breast cancer cells (i.e., SKBR3) that overexpress HER2 receptors for the inhibition of cancer cell growth under 808 nm laser irradiation. This study highlights a facile and controllable method to fabricate therapeutic nanoparticles capable of targeted delivery.

Synthesis, characterisation and evaluation of hyperbranched N-(2-hydroxypropyl) methacrylamides for transport and delivery in pancreatic cell lines in vitro and in vivo.

Hyperbranched polymers have many promising features for drug delivery, owing to their ease of synthesis, multiple functional group content, and potential for high drug loading with retention of solubility. Here we prepared hyperbranched N-(2-hydroxypropyl)methacrylamide (HPMA) polymers with a range of molar masses and particle sizes, and with attached dyes, radiolabel or the anticancer drug gemcitabine. Reversible addition-fragmentation chain transfer (RAFT) polymerisation enabled the synthesis of pHPMA polymers and a gemcitabine-comonomer functionalised pHPMA polymer pro-drug, with diameters of the polymer particles ranging from 7-40 nm. The non-drug loaded polymers were well-tolerated in cancer cell lines and macrophages, and were rapidly internalised in 2D cell culture and transported efficiently to the centre of dense pancreatic cancer 3D spheroids. The gemcitabine-loaded polymer pro-drug was found to be toxic both to 2D cultures of MIA PaCa-2 cells and also in reducing the volume of MIA PaCa-2 spheroids. The non-drug loaded polymers caused no short-term adverse effects in healthy mice following systemic injection, and derivatives of these polymers labelled with 89Zr-were tracked for their distribution in the organs of healthy and MIA PaCa-2 xenograft bearing Balb/c nude mice. Tumour accumulation, although variable across the samples, was highest in individual animals for the pHPMA polymer of ∼20 nm size, and accordingly a gemcitabine pHPMA polymer pro-drug of ∼18 nm diameter was evaluated for efficacy in the tumour-bearing animals. The efficacy of the pHPMA polymer pro-drug was very similar to that of free gemcitabine in terms of tumour growth retardation, and although there was a survival benefit after 70 days for the polymer pro-drug, there was no difference at day 80. These data suggest that while polymer pro-drugs of this type can be effective, better tumour targeting and enhanced in situ release remain as key obstacles to clinical translation even for relatively simple polymers such as pHPMA.