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.

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.

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.

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.

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.

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.

Antibody-Based Formats to Target Glioblastoma: Overcoming Barriers to Protein Drug Delivery.

Glioblastoma (GB) is recognized as the most aggressive form of primary brain cancer. Despite advances in treatment strategies that include surgery, radiation, and chemotherapy, the median survival time (∼15 months) of patients with GB has not significantly improved. The poor prognosis of GB is also associated with a very high chance of tumor recurrence (∼90%), and current treatment measures have failed to address the complications associated with this disease. However, targeted therapies enabled through antibody engineering have shown promise in countering GB when used in combination with conventional approaches. Here, we discuss the challenges in conventional as well as future GB therapeutics and highlight some of the known advantages of using targeted biologics to overcome these impediments. We also review a broad range of potential alternative routes that could be used clinically to administer anti-GB biologics to the brain through evasion of its natural barriers.

Engineering Fluorescent Gold Nanoclusters Using Xanthate-Functionalized Hydrophilic Polymers: Toward Enhanced Monodispersity and Stability.

We introduce xanthate-functionalized poly(cyclic imino ethers)s (PCIEs), specifically poly(2-ethyl-2-oxazoline) and poly(2-ethyl-2-oxazine) given their stealth characteristics, as an attractive alternative to conventional thiol-based ligands for the synthesis of highly monodisperse and fluorescent gold nanoclusters (AuNCs). The xanthate in the PCIEs interacts with Au ions, acting as a well-controlled template for the direct formation of PCIE-AuNCs. This method yields red-emitting AuNCs with a narrow emission peak (λem = 645 nm), good quantum yield (4.3-4.8%), long fluorescence decay time (∼722-844 ns), and unprecedented product yield (>98%). The PCIE-AuNCs exhibit long-term colloidal stability, biocompatibility, and antifouling properties, enabling a prolonged blood circulation, lower nonspecific accumulation in major organs, and better renal clearance when compared with AuNCs without polymer coating. The advances made here in the synthesis of metal nanoclusters using xanthate-functionalized PCIEs could propel the production of highly monodisperse, biocompatible, and renally clearable nanoprobes in large-scale for different theranostic applications.

Hyperbranched Poly(2-oxazoline)s and Poly(ethylene glycol): A Structure-Activity Comparison of Biodistribution.

In light of research reporting abnormal pharmacokinetic behavior for therapeutics and formulations containing poly(ethylene glycol) (PEG), a renewed emphasis has been placed on exploring alternative surrogate materials and tailoring specific materials to distinct nanomedicine applications. Poly(2-oxazolines) (POx) have shown great promise in this regard; however, a comparison of POx and PEG interactions with components of the immune system is needed to inform on their distinct suitability. Herein, the interaction of isolated immune cells following injection of hyperbranched polymers comprised of PEG or hydrophilic POx macromonomers was determined via flow cytometry. All materials showed similar association with all of the splenic immune cells analyzed. Interestingly, splenic CD68hi and CD11bhi macrophages showed similar levels of polymer association, despite CD11bhi being a smaller population, suggesting CD68 is linked to increased recognition and phagocytosis of these nanomaterials. This is of interest given that CD68 is a scavenger receptor and directly facilitates the clearance of cellular debris and promotion of phagocytosis, as opposed to CD11b, which is associated with the mediating inflammation via the production of cytokines as well as complement-mediated uptake of foreign particles. In the liver, PEG and poly(2-methyl oxazoline) hyperbranched polymers showed no discernible differences in their cellular association, while hyperbranched poly(2-ethyl oxazoline) showed increased association with dendrocytes and CD68hi macrophages, suggesting that this material exhibited a greater propensity to interact with components of the immune system. This work highlights the importance of how subtle changes in chemical structure can influence the immune response.

Direct Comparison of Poly(ethylene glycol) and Phosphorylcholine Drug-Loaded Nanoparticles In Vitro and In Vivo.

Phosphorylcholine is known to repel the absorption of proteins onto surfaces, which can prevent the formation of a protein corona on the surface of nanoparticles. This can influence the fate of nanoparticles used for drug delivery. This material could therefore serve as an alternative to poly(ethylene glycol) (PEG). Herein, the synthesis of different particles prepared by polymerization-induced self-assembly (PISA) coated with either poly(ethylene glycol) (PEG) or zwitterionic 2-methacryloyloxyethyl phosphorylcholine (MPC) and 4-(N-(S-penicillaminylacetyl)amino) phenylarsenonous acid (PENAO) was reported. The anticancer drug 4-(N-(S-penicillaminylacetyl)amino) phenylarsenonous acid (PENAO) was conjugated to the shell-forming block. Interactions of the different coated nanoparticles, which present comparable sizes and size distributions (76-85 nm, PDI = 0.067-0.094), with two-dimensional (2D) and three-dimensional (3D) cultured cells were studied, and their cytotoxicities, cellular uptakes, spheroid penetration, and cell localization profiles were analyzed. While only a minimal difference in behaviour was observed for nanoparticles assessed using in vitro experiment (with PEG-co- PENAO-coated micelles showing slightly higher cytotoxicity and better spheroid penetration and cell localization ability), the effect of the different physicochemical properties between nanoparticles had a more dramatic effect on in vivo biodistribution. After 1 h of injection, the majority of the MPC-co-PENAO-coated nanoparticles were found to accumulate in the liver, making this particle system unfeasible for future biological studies.

Next-Generation Polymeric Nanomedicines for Oncology: Perspectives and Future Directions.

Precision polymers as advanced nanomedicines represent an appealing approach for the treatment of otherwise untreatable malignancies. By taking advantage of unique nanomaterial properties and implementing judicious design strategies, polymeric nanomedicines are able to be produced that overcome many barriers to effective treatment. Current key research focus areas anticipated to produce the greatest impact in polymer applications in nanomedicine for oncology include new strategies to achieve "active" targeting, polymeric pro-drug activation, and combinatorial polymer drug delivery approaches in combination with enhanced understanding of complex bio-nano interactions. These approaches, both in isolation or combination, form the next generation of precision nanomedicines with significant anticipated future health outcomes. Of necessity, these approaches will combine an intimate understanding of biological interactions with advanced materials design. This perspectives piece aims to highlight emerging opportunities that promise to be game changers in the nanomedicine oncology field. Discussed herein are current and next generation polymeric nanomedicines with a focus towards structures that are, or could, undergo clinical translation as well as highlight key advances in the field.

Effect of Chain-End Chemistries on the Efficiency of Coupling Antibodies to Polymers Using Unnatural Amino Acids.

Novel conjugates that incorporate strategies for increasing the therapeutic payload, such as targeted polymeric delivery vehicles, have great potential in overcoming limitations of conventional antibody therapies that often exhibit immunogenicity and limited drug loading. Click chemistry has significantly expanded the toolbox of effective strategies for developing hybrid polymer-biomolecule conjugates, however, effective systems require orthogonality between the polymer and biomolecule chemistries to achieve efficient coupling. Here, three cycloaddition-based strategies for antibody conjugation to polymeric carriers are explored and show that a purely radical-based method for polymer synthesis and subsequent biomolecule attachment has a trade-off between coupling efficiency of the antibody and the ability to synthesize polymers with controlled chemical properties. It is shown that careful consideration of both coupling chemistries as well as the potential effect of how this modulates the chemical properties of the polymer nanocarrier should be considered during the development of such systems. The strategies described offer insight into improving conjugate development for therapeutic and theranostic applications. In this system, polymerization using conventional and established reversible addition fragmentation chain transfer (RAFT) agents, followed by multiple post-modification steps, always leads to systems with more defined chemical architectures compared to strategies that utilize alkyne-functional RAFT agents.

Using Peptide Aptamer Targeted Polymers as a Model Nanomedicine for Investigating Drug Distribution in Cancer Nanotheranostics.

Theranostics is a strategy that combines multiple functions such as targeting, stimulus-responsive drug release, and diagnostic imaging into a single platform, often with the aim of developing personalized medicine.1,2 Based on this concept, several well-established hyperbranched polymeric theranostic nanoparticles were synthesized and characterized as model nanomedicines to investigate how their properties affect the distribution of loaded drugs at both the cell and whole animal levels. An 8-mer peptide aptamer was covalently bound to the periphery of the nanoparticles to achieve both targeting and potential chemosensitization functionality against heat shock protein 70 (Hsp70). Doxorubicin was also bound to the polymeric carrier as a model chemotherapeutic drug through a degradable hydrazone bond, enabling pH-controlled release under the mildly acid conditions that are found in the intracellular compartments of tumor cells. In order to track the nanoparticles, cyanine-5 (Cy5) was incorporated into the polymer as an optical imaging agent. In vitro cellular uptake was assessed for the hyperbranched polymer containing both doxorubicin (DOX) and Hsp70 targeted peptide aptamer in live MDA-MB-468 cells, and was found to be greater than that of either the untargeted, DOX-loaded polymer or polymer alone due to the specific affinity of the peptide aptamer for the breast cancer cells. This was also validated in vivo with the targeted polymers showing much higher accumulation within the tumor 48 h postinjection than the untargeted analogue. More detailed assessment of the nanomedicine distribution was achieved by directly following the polymeric carrier and the doxorubicin at both the in vitro cellular level via compartmental analysis of confocal images of live cells and in whole tumors ex vivo using confocal imaging to visualize the distribution of the drug in tumor tissue as a function of distance from blood vessels. Our results indicate that this polymeric carrier shows promise as a cancer theranostic, demonstrating active targeting to tumor cells with the capability for simultaneous drug release.

Targeting Nanomedicines to Prostate Cancer: Evaluation of Specificity of Ligands to Two Different Receptors In Vivo.

PURPOSE: This manuscript utilised in vivo multispectral imaging to demonstrate the efficacy of two different nanomedicine formulations for targeting prostate cancer. METHODS: Pegylated hyperbranched polymers were labelled with fluorescent markers and targeting ligands against two different prostate cancer markers; prostate specific membrane antigen (PSMA) and the protein kinase, EphrinA2 receptor (EphA2). The PSMA targeted nanomedicine utilised a small molecule glutamate urea inhibitor of the protein, while the EphA2 targeted nanomedicine was conjugated to a single-chain variable fragment based on the antibody 4B3 that has shown high affinity to the receptor. RESULTS: Hyperbranched polymers were synthesised bearing the different targeting ligands. In the case of the EphA2-targeting nanomedicine, significant in vitro uptake was observed in PC3 prostate cancer cells that overexpress the receptor, while low uptake was observed in LNCaP cells (that have minimal expression of this receptor). Conversely, the PSMA-targeted nanomedicine showed high uptake in LNCaP cells, with only minor uptake in the PC3 cells. In a dual-tumour xenograft mouse model, the nanomedicines showed high uptake in tumours in which the receptor was overexpressed, with only minimal non-specific accumulation in the low-expression tumours. CONCLUSIONS: This work highlighted the importance of clearly defining the target of interest in next-generation nanomedicines, and suggests that dual-targeting in such nanomedicines may be a means to achieve greater efficacy.

Bioproduction of highly charged designer peptide surfactants via a chemically cleavable coiled-coil heteroconcatemer.

Designer peptides have recently attracted attention as self-assembling fibrils, hydrogelators and green surfactants with the potential for sustainable bioproduction. Carboxylate-rich peptides in particular have shown potential as salt-resistant emulsifiers; however the expression of highly charged peptides of this kind remains a challenge. To achieve expression of a strongly anionic helical surfactant peptide, we paired the peptide with a cationic helical partner in a coiled-coil miniprotein and optimized the polypeptide sequence for net charge, hydropathy and predicted protease resistance (via the Guruprasad instability index). Our design permitted expression of a soluble concatemer that accumulates to high levels (22% of total protein) in E. coli. The concatemer showed high stability to heat and proteases, allowing isolation by simple heat and pH precipitation steps that yield concatemer at 133 mg per gram of dry cell weight and >99% purity. Aspartate-proline sites were included in the concatemer to allow cleavage with heat and acid to give monomeric peptides. We characterized the acid cleavage pathway of the concatemer by coupled liquid chromatography-mass spectrometry and modeled the kinetic pathways involved. The outcome represents the first detailed kinetic characterization of protein cleavage at aspartate-proline sites, and reveals unexpected cleavage preferences, such as favored cleavage at the C-termini of peptide helices. Chemical denaturation of the concatemer showed an extremely high thermodynamic stability of 38.9 kcal mol(-1) , with cleavage decreasing the stability of the coiled coil to 32.8 kcal mol(-1) . We determined an interfacial pressure of 29 mN m(-1) for both intact and cleaved concatemer at the air-water interface, although adsorption was slightly more rapid for the cleaved peptides. The cleaved peptides could be used to prepare heat-stable emulsions with droplet sizes in the nanometer range.

Evaluation of Polymeric Nanomedicines Targeted to PSMA: Effect of Ligand on Targeting Efficiency.

Targeted nanomedicines offer a strategy for greatly enhancing accumulation of a therapeutic within a specific tissue in animals. In this study, we report on the comparative targeting efficiency toward prostate-specific membrane antigen (PSMA) of a number of different ligands that are covalently attached by the same chemistry to a polymeric nanocarrier. The targeting ligands included a small molecule (glutamate urea), a peptide ligand, and a monoclonal antibody (J591). A hyperbranched polymer (HBP) was utilized as the nanocarrier and contained a fluorophore for tracking/analysis, whereas the pendant functional chain-ends provided a handle for ligand conjugation. Targeting efficiency of each ligand was assessed in vitro using flow cytometry and confocal microscopy to compare degree of binding and internalization of the HBPs by human prostate cancer (PCa) cell lines with different PSMA expression status (PC3-PIP (PSMA+) and PC3-FLU (PSMA-). The peptide ligand was further investigated in vivo, in which BALB/c nude mice bearing subcutaneous PC3-PIP and PC3-FLU PCa tumors were injected intravenously with the HBP-peptide conjugate and assessed by fluorescence imaging. Enhanced accumulation in the tumor tissue of PC3-PIP compared to PC3-FLU highlighted the applicability of this system as a future imaging and therapeutic delivery vehicle.

Mitigating the Effects of Persistent Antipolymer Immune Reactions in Nanomedicine: Evaluating Materials-Based Approaches Using Molecular Imaging.

Poly(ethylene glycol) (PEG) is a hydrophilic polymer ubiquitously used in both medical and nonmedical goods. Recent debate surrounding the observed stimulation of immune responses against PEG has spurred the development of materials that may be suitable replacements for this common polymeric component. The underlying view is that these alternative materials with comparable physicochemical properties can overcome the unfavorable and unpredictable effects of antibody-mediated clearance by being chemically, and therefore antigenically, distinct from PEG. However, this hypothesis has not been thoroughly tested in any defined manner, and the immune response observed against PEG has not been rigorously investigated within the context of these emerging materials. Consequently, it remains unclear whether immunity-mediated discrimination between polymeric entities even occurs in vivo and, if this is the case, how it may be exploited. In this study, we utilize positron emission tomography-computed tomography molecular imaging in mice immunized to develop specific antibody responses to PEG and an alternative polymer in order to visualize and quantify the influence of antipolymer antibodies on the biodistribution of synthetic polymers in vivo as a function of immunization status. Under the conditions of this experiment, mice could be primed to exhibit both innate and adaptive immunity to all of the polymer systems to which they were exposed. We demonstrate that alternating between chemically disparate polymers is a viable approach to extend their efficacy when antipolymer humoral immune responses arise.

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.

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.