Engineering immunomodulatory nanoplatforms from commensal bacteria-derived polysaccharide A.

Capsular zwitterionic polysaccharides (CZPs), typically found on the surfaces of commensal gut bacteria, are important immunomodulatory molecules due to their ability to produce a T cell dependent immune response upon processing by antigen presenting cells (APCs). Their immunological activity makes them potentially useful for generating material constructs that are applicable for the treatment of diseases, or as vaccines. Herein, we explored synthetic strategies to generate immunologically active polymer-carbohydrate conjugates and nanomaterials of the CZP, Polysaccharide A (PSA) derived from Bacteroides fragilis. Initially, we addressed the purification of PSA, which is critical for the realization of materials applicable for biomedical purposes. Anion exchange high performance liquid chromatography in the presence of a surfactant (CHAPS) enabled the isolation of pure PSA. Through modification of purified PSA with azide groups, we demonstrated that polymers or antigens could be incorporated with PSA via click chemistry reactions to generate conjugates that can be fabricated into nanoparticles. By conjugation of PSA with a DBCO end functionalized polyphosphoester polymer with hydrophobic pendant terminal alkyne groups, an amphiphilic conjugate was obtained which formed nanoparticles of about 100 nm in aqueous solution. Moreover, terminal alkyne groups could be modified with charged thiol molecules (amine/carboxylate) via thiol-yne radical chemistry to generate conjugates, which could be incorporated into nanoparticles via electrostatic interactions building onto a charged nanoparticle template. The conjugates and nanoparticles exhibited immunological activity as assessed by the toll-like receptor 2 (TLR2) activation assay and positive cytokine production (IL-10) following their co-incubation with APCs and T cells. Summarily, this work plainly demonstrates chemical biology strategies for fabricating immunomodulatory nanomaterials from commensal microorganisms that can potentially be novel vaccines or immunotherapeutics.

The roadmap to micro: Generation of micron-sized polymeric particles using a commercial microfluidic system.

Microfluidic-assisted particle fabrication provides a route to circumvent the disadvantages associated with traditional methods of polymeric particle generation, such as low drug loading efficiency, challenges in controlling encapsulated drug release rates, batch-to-batch variability in particle physical properties and formulation instability. However, this approach primarily produces particles with nanometer size dimensions, which limits drug delivery modalities. Herein, we systematically studied parameters for the generation of micron-sized poly(lactic-co-glycolic) acid (PLGA) particles using a microfluidic system, the NanoAssemblr benchtop. Initially, we used two organic solvents that have been reported suitable for the fabrication of PLGA nanoparticles - acetone and acetonitrile. Subsequently, we methodically manipulated polymer concentration, organic: aqueous flow rate ratios, total flow rate, organic phase composition, and surfactant concentration to develop a route for the fabrication of micron-sized PLGA particles. Further, we incorporated hydroxychloroquine (HCQ), a clinically approved drug for malaria and lymphoma, and measured how its incorporation impacted particle physicochemical properties. Briefly, altering the organic phase composition by including ethyl acetate (less polar solvent), resulted in micron-scale particles, as well as increased polydispersity indexes (PDIs). Adjusting the surfactant concentration of poly vinyl alcohol (PVA) after the addition of these solvent mixtures rendered large particles with lower PDI variability. Moreover, encapsulation of HCQ influenced particle hydrodynamic diameter and PDI in a PVA concentration dependent manner. Finally, we demonstrated that unloaded and HCQ-loaded microparticles did not affect the viability of RAW 264.7 macrophages. This study provides an itinerary for fabricating biocompatible, drug-loaded, micron-sized polymeric particles, particularly when the drug of interest is not readily soluble in conventional organic solvents.

Systemic Immunotherapy with Micellar Resiquimod-Polymer Conjugates Triggers a Robust Antitumor Response in a Breast Cancer Model.

Resiquimod is an immunopotent toll-like receptor 7/8 agonist with antitumor activity. Despite being potent against skin cancers, it is poorly tolerated systemically due to toxicity. Integrating resiquimod into nanoparticles presents an avenue to circumvent the toxicity problem. Herein, the preparation of degradable nanoparticles with covalently bound resiquimod and their systemic application in cancer immunotherapy is reported. Dispersion in water of amphiphilic constructs integrating resiquimod covalently bound via degradable amide or ester linkages yields immune-activating nanoparticles. The degradable agonist-nanoparticle bonds allow the release of resiquimod from the carrier nanoparticles. In vitro assays with antigen presenting cells demonstrate that the nanoparticles retain the immunostimulatory activity of resiquimod. Systemic administration of the nanoparticles and checkpoint blockade (aPD-1) to a breast cancer mouse model with multiple established tumors triggers antitumor activity evidenced by suppressed tumor growth and enhanced CD8+ T-cell infiltration. Nanoparticles with ester links, which hydrolyze more readily, yield a stronger immune response with 75% of tumors eliminated when combined with aPD-1. The reduced tumor growth and the presence of activated CD8+ T-cells across multiple tumors suggest the potential for treating metastatic cancer.

Gemcitabine-retinoid prodrug loaded nanoparticles display in vitro antitumor efficacy towards drug-resilient human PANC-1 pancreatic cancer cells.

The treatment of pancreatic cancer with gemcitabine is hampered by its rapid metabolism in vivo, the dense stroma around the tumor site which prevents the drug from reaching the cancerous cells and drug resistance. To address these challenges, this study describes the preparation of a retinoid prodrug of gemcitabine, GemRA (gemcitabine conjugated to retinoic acid), and its formulation into a nanoparticulate system applicable for pancreatic cancer treatment. Retinoic acid targets stellate cells which are part of the stroma and can thus augment the delivery of gemcitabine. GemRA dissolved in dimethylsulfoxide presented efficacy towards PANC-1 (human) and mT4 (mouse) pancreatic cancer cell lines but its poor solubility in aqueous solution affects its applicability. Thus, the preparation of the nanoparticles was initially attempted through self-assembly of GemRA, which resulted in the formation of unstable aggregates that precipitated during preparation. As a result, encapsulation of the drug into micelles of polyethylene glycol-retinoic acid (PGRA) amphiphilic conjugates was accomplished and resulted in successful incorporation of GemRA into nanoparticles of ca. 33 nm by dynamic light scattering and 25 nm by transmission electron microscopy. The nanoparticles had good stability in aqueous media and protected gemcitabine from the enzymatic action of cytidine deaminase, which converts gemcitabine to its inactive metabolite upon circulation. Cellular uptake of the nanoparticles by PANC-1 cells was confirmed by fluorescence spectroscopy and flow cytometry. Treatment of PANC-1 cells in vitro with the prodrug-loaded nanoparticles resulted in a significant reduction in cell viability (IC50 ca. 5 µM) compared to treatment with gemcitabine (IC50 > 1000 µM). The ability of the GemRA-loaded nanoparticles to induce cellular apoptosis of treated PANC-1 cells was ascertained via a TUNEL assay suggesting these nanoparticles are effective in pancreatic cancer treatment.

Thermoresponsive Iron Oxide Nanocubes for an Effective Clinical Translation of Magnetic Hyperthermia and Heat-Mediated Chemotherapy.

The use of magnetic nanoparticles in oncothermia has been investigated for decades, but an effective combination of magnetic nanoparticles and localized chemotherapy under clinical magnetic hyperthermia (MH) conditions calls for novel platforms. In this study, we have engineered magnetic thermoresponsive iron oxide nanocubes (TR-cubes) to merge MH treatment with heat-mediated drug delivery, having in mind the clinical translation of the nanoplatform. We have chosen iron oxide based nanoparticles with a cubic shape because of their outstanding heat performance under MH clinical conditions, which makes them benchmark agents for MH. Accomplishing a surface-initiated polymerization of strongly interactive nanoparticles such as our iron oxide nanocubes, however, remains the main challenge to overcome. Here, we demonstrate that it is possible to accelerate the growth of a polymer shell on each nanocube by simple irradiation of a copper-mediated polymerization with a ultraviolet light (UV) light, which both speeds up the polymerization and prevents nanocube aggregation. Moreover, we demonstrate herein that these TR-cubes can carry chemotherapeutic doxorubicin (DOXO-loaded-TR-cubes) without compromising their thermoresponsiveness both in vitro and in vivo. In vivo efficacy studies showed complete tumor suppression and the highest survival rate for animals that had been treated with DOXO-loaded-TR-cubes, only when they were exposed to MH. The biodistribution of intravenously injected TR-cubes showed signs of renal clearance within 1 week and complete clearance after 5 months. This biomedical platform works under clinical MH conditions and at a low iron dosage, which will enable the translation of dual MH/heat-mediated chemotherapy, thus overcoming the clinical limitation of MH: i.e., being able to monitor tumor progression post-MH-treatment by magnetic resonance imaging (MRI).

A Scalable Method for Squalenoylation and Assembly of Multifunctional 64Cu-Labeled Squalenoylated Gemcitabine Nanoparticles.

Squalenoylation of gemcitabine, a front-line therapy for pancreatic cancer, allows for improved cellular-level and system-wide drug delivery. The established methods to conjugate squalene to gemcitabine and to form nanoparticles (NPs) with the squalenoylated gemcitabine (SqGem) conjugate are cumbersome, time-consuming and can be difficult to reliably replicate. Further, the creation of multi-functional SqGem-based NP theranostics would facilitate characterization of in vivo pharmacokinetics and efficacy. Methods: Squalenoylation conjugation chemistry was enhanced to improve reliability and scalability using tert-butyldimethylsilyl (TBDMS) protecting groups. We then optimized a scalable microfluidic mixing platform to produce SqGem-based NPs and evaluated the stability and morphology of select NP formulations using dynamic light scattering (DLS) and transmission electron microscopy (TEM). Cytotoxicity was evaluated in both PANC-1 and KPC (KrasLSL-G12D/+; Trp53LSL-R172H/+; Pdx-Cre) pancreatic cancer cell lines. A 64Cu chelator (2-S-(4-aminobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid, NOTA) was squalenoylated and used with positron emission tomography (PET) imaging to monitor the in vivo fate of SqGem-based NPs. Results: Squalenoylation yields of gemcitabine increased from 15% to 63%. Cholesterol-PEG-2k inclusion was required to form SqGem-based NPs using our technique, and additional cholesterol inclusion increased particle stability at room temperature; after 1 week the PDI of SqGem NPs with cholesterol was ~ 0.2 while the PDI of SqGem NPs lacking cholesterol was ~ 0.5. Similar or superior cytotoxicity was achieved for SqGem-based NPs compared to gemcitabine or Abraxane® when evaluated at a concentration of 10 µM. Squalenoylation of NOTA enabled in vivo monitoring of SqGem-based NP pharmacokinetics and biodistribution. Conclusion: We present a scalable technique for fabricating efficacious squalenoylated-gemcitabine nanoparticles and confirm their pharmacokinetic profile using a novel multifunctional 64Cu-SqNOTA-SqGem NP.

Unimicellar hyperstars as multi-antigen cancer nanovaccines displaying clustered epitopes of immunostimulating peptides.

Unimicellar hyperstar macromolecular chimeras displaying multiple melanoma peptide antigens were prepared primarily via a combination of click chemistry and esterification reactions starting from a biodegradable hyperbranched polymer template. Solubilization of the hyperstars in aqueous solution afforded a multi-antigen unimicellar cancer nanovaccine of about 20 nm. The nanovaccine showed good biocompatibility and uptake by dendritic cells in vitro. An in vivo evaluation of the nanovaccine therapeutic efficacy against melanoma in mice implanted with B16OVA tumors revealed significantly greater T-cell recruitment and improved survival rates for mice treated with nanovaccine and adjuvant compared to non-treated mice.

Dually responsive gold-iron oxide heterodimers: merging stimuli-responsive surface properties with intrinsic inorganic material features.

We demonstrate a versatile approach for the preparation of dually responsive smart inorganic heterostructures (HSs) with the potential for exploitation in nanomedicine. We utilize Au-FexOy dimers as templates for generating smart inorganic HSs with a pH-responsive coating and a thermo-responsive coating attached to iron oxide and gold nanoparticles (NPs), respectively. First, a thiol-modified thermo-responsive (PNIPAAM-co-PEGA) polymer could be selectively attached to the gold domain by ligand exchange. The sequential attachment of a catechol-modified initiator to the iron oxide surface enables the in situ polymerization of a pH-responsive (PDMAEA) polymer. As hereby shown, the presence of the two distinct polymer domains on each NP subdomain enables each side of the HS to be loaded with different agents. Indeed, by a gel electrophoresis experiment we demonstrate the loading of siRNA on the pH-responsive polymer and the loading of Nile Blue dye, used as a drug model molecule, on the thermo-responsive polymer. The smart HSs exhibited good biocompatibility and downregulated GFP production when loaded with anti-GFP siRNA molecules. In addition, an investigation of the magnetic relaxivity times revealed that the high R2 relaxivity values of the HSs suggest their potential as contrast agents in magnetic resonance imaging (MRI) applications.

Toward Personalized Peptide-Based Cancer Nanovaccines: A Facile and Versatile Synthetic Approach.

Personalized cancer vaccines (PCVs) are receiving attention as an avenue for cancer immunotherapy. PCVs employ immunogenic peptide epitopes capable of stimulating the immune system to destroy cancer cells with great specificity. Challenges associated with effective delivery of these peptides include poor solubility of hydrophobic sequences, rapid clearance, and poor immunogenicity, among others. The incorporation of peptides into nanoparticles has the potential to overcome these challenges, but the broad range of functionalities found in amino acids presents a challenge to conjugation due to possible interferences and lack of reaction specificity. Herein, a facile and versatile approach to generating nanosized PCVs under mild nonstringent conditions is reported. Following a simple two-step semibatch synthetic approach, amphiphilic hyperbranched polymer-peptide conjugates were prepared by the conjugation of melanoma antigen peptides, either TRP2 (hydrophobic) or MUT30 (hydrophilic), to an alkyne functionalized core via strain-promoted azide-alkyne click chemistry. Self-assembly of the amphiphiles gave spherical nanovaccines (by transmission electron microscopy) with sizes in the range of 10-30 nm (by dynamic light scattering). Fluorescently labeled nanovaccines were prepared to investigate the cellular uptake by antigen presenting cells (dendritic cells), and uptake was confirmed by flow cytometry and microscopy. The TRP2 nanovaccine was taken up the most followed by MUT30 nanoparticles and, finally, nanoparticles without peptide. The nanovaccines showed good biocompatibility against B16-F10 cells, yet the TRP2 peptide showed signs of toxicity, possibly due to its hydrophobicity. A test for immunogenicity revealed that the nanovaccines were poorly immunogenic, implying the need for an adjuvant when administered in vivo. Treatment of mice with melanoma tumors showed that in combination with adjuvant, CpG, groups with the peptide nanovaccines slowed tumor growth and improved survival (up to 24 days, TRP2) compared to the untreated group (14 days).

Functionalization of strongly interacting magnetic nanocubes with (thermo)responsive coating and their application in hyperthermia and heat-triggered drug delivery.

Herein, we prepare nanohybrids by incorporating iron oxide nanocubes (cubic-IONPs) within a thermoresponsive polymer shell that can act as drug carriers for doxorubicin(doxo). The cubic-shaped nanoparticles employed are at the interface between superparamagnetic and ferromagnetic behavior and have an exceptionally high specific absorption rate (SAR), but their functionalization is extremely challenging compared to bare superparamagnetic iron oxide nanoparticles as they strongly interact with each other. By conducting the polymer grafting reaction using reversible addition-fragmentation chain transfer (RAFT) polymerization in a viscous solvent medium, we have here developed a facile approach to decorate the nanocubes with stimuli-responsive polymers. When the thermoresponsive shell is composed of poly(N-isopropylacrylamide-co-polyethylene glycolmethyl ether acrylate), nanohybrids have a phase transition temperature, the lower critical solution temperature (LCST), above 37 °C in physiological conditions. Doxo loaded nanohybrids exhibited a negligible drug release below 37 °C but showed a consistent release of their cargo on demand by exploiting the capability of the nanocubes to generate heat under an alternating magnetic field (AMF). Moreover, the drug free nanocarrier does not exhibit cytotoxicity even when administered at high concentration of nanocubes (1g/L of iron) and internalized at high extent (260 pg of iron per cell). We have also implemented the synthesis protocol to decorate the surface of nanocubes with poly(vinylpyridine) polymer and thus prepare pH-responsive shell coated nanocubes.

Polymer coated inorganic nanoparticles: tailoring the nanocrystal surface for designing nanoprobes with biological implications.

The use of inorganic nanoparticles in biomedicine, in particular in the field of diagnosis and therapy of human diseases, has rapidly grown in the last few decades. Water solubilisation of the nanoparticles, especially for particles synthesized in non-polar solvents, is an essential prerequisite for their biological exploitation. The encapsulation of surfactant coated nanoparticles into polymer shells represents one of the most suitable and most popular methods to make them water soluble. Herein we provide an overview of the amphiphilic polymer molecules used and the efforts undertaken to further tailor the surface of polymer coated nanoparticles with fluorescent dyes, chemical sensor molecules and small or large biomolecules for the preparation of bio-functional nanoprobes. Their biological implications, highlighting limitations and challenges, are also discussed.

Polymer-peptide chimeras for the multivalent display of immunogenic peptides.

Chimeras of poly(n-isopropyl acrylamide) and immunogenic peptides from the cancer-associated glycoprotein MUC1 were synthesised using a combination of solid-phase peptide synthesis, RAFT polymerisation and copper-catalysed alkyne-azide cycloaddition reactions.

Orthogonal "relay" reactions for designing functionalized soft nanoparticles.

The combination of reversible addition-fragmentation chain transfer (RAFT) chemistry followed by thiol-based "click" chemistry, known as an orthogonal "relay" reaction as one step complements the other, was used to produce surface-functionalized soft nanoparticles. Thiocarbonyl thio compounds were first used in the presence of vinyl monomers and a source of radicals to control the growth of the polymeric chains (via the RAFT process) and then reduced to thiols and utilized as a handle for functionalization of the resulting polymer chain ends (via a thiol-based click reaction). Both reactions occur under mild conditions and offer excellent control over the properties of the final product, and the thiol addition shows all the benefits of a click reaction, without requiring the use of a catalyst. This simple chemistry opens up the route to the production of a wide range of functional materials, and the concept is illustrated by the formation of nanoparticle-based gels, fluorescent-tagged particles, and protein-nanoparticle conjugates.