Fully synthetic injectable depots with high drug content and tunable pharmacokinetics for long-acting drug delivery.

Clinical studies have validated that antiretroviral (ARV) drugs can serve as an HIV pre-exposure prophylactic (PrEP) strategy. Dosing adherence remains a crucial factor determining the final efficacy outcomes, and both long-acting implants and injectable depot systems are being developed to improve patient adherence. Here, we describe an injectable depot platform that exploits a new mechanism for both formation and controlled release. The depot is a polymeric prodrug synthesized from monomers that incorporate an ARV drug tenofovir alafenamide (TAF) with degradable linkers that can be designed to control release rates. The prodrug monomers are synthetically incorporated into homopolymer or block designs that exhibit high drug weight percent (wt%) and also are hydrophobized in these prodrug segments to drive depot formation upon injection. Drug release converts those monomers to more hydrophilic pendant groups via linker cleavage, and as this drug release proceeds, the polymer chains losing hydrophobicity are then disassociated from the depot and released over time to provide a depot dissolution mechanism. We show that long-acting TAF depots can be designed as block copolymers or as homopolymers. They can also be designed with different linkers, for example with faster or slower degrading p-hydroxybenzyloxycarbonyl (Benzyl) and ethyloxycarbonyl (Alkyl) linkers, respectively. Diblock designs of p(glycerol monomethacrylate)-b-p(Alkyl-TAF-methacrylate) and p(glycerol monomethacrylate)-b-p(Benzyl-TAF-methacrylate) were first characterized in a mouse subcutaneous injection model. The alkylcarbamate linker design (TAF 51 wt%) showed excellent sustained release profiles of the key metabolite tenofovir (TFV) in skin and plasma over a 50-day period. Next, the homopolymer design with a high TAF drug wt% of 73% was characterized in the same model. The homopolymer depots with p(Alkyl-TAFMA) exhibited sustained TFV and TAF release profiles in skin and blood over 60 days, and TFV-DP concentrations in peripheral blood mononuclear cells (PBMC) were found to be at least 10-fold higher than the clinically suggested minimally EC90 protective concentration of 24 fmol/106 cells. These are the first reports of sustained parent TAF dosing observed in mouse and TFV-DP in mouse PBMC. IVIS imaging of rhodamine labeled homopolymer depots showed that degradation and release of the depot coincided with the sustained TAF release. Finally, these polymers showed excellent stability in accelerated stability studies over a six-month time period, and exceptional solubility of over 700 mg/mL in the DMSO formulation solvent. The homopolymer designs have a drug reservoir potential of well over a year at mg/day dosing and may not require cold chain storage for global health and developed world long-acting drug delivery applications.

An ultrafast insulin formulation enabled by high-throughput screening of engineered polymeric excipients.

Insulin has been used to treat diabetes for almost 100 years; yet, current rapid-acting insulin formulations do not have sufficiently fast pharmacokinetics to maintain tight glycemic control at mealtimes. Dissociation of the insulin hexamer, the primary association state of insulin in rapid-acting formulations, is the rate-limiting step that leads to delayed onset and extended duration of action. A formulation of insulin monomers would more closely mimic endogenous postprandial insulin secretion, but monomeric insulin is unstable in solution using present formulation strategies and rapidly aggregates into amyloid fibrils. Here, we implement high-throughput-controlled radical polymerization techniques to generate a large library of acrylamide carrier/dopant copolymer (AC/DC) excipients designed to reduce insulin aggregation. Our top-performing AC/DC excipient candidate enabled the development of an ultrafast-absorbing insulin lispro (UFAL) formulation, which remains stable under stressed aging conditions for 25 ± 1 hours compared to 5 ± 2 hours for commercial fast-acting insulin lispro formulations (Humalog). In a porcine model of insulin-deficient diabetes, UFAL exhibited peak action at 9 ± 4 min, whereas commercial Humalog exhibited peak action at 25 ± 10 min. These ultrafast kinetics make UFAL a promising candidate for improving glucose control and reducing burden for patients with diabetes.

Bacterial membrane permeability of antimicrobial polymethacrylates: Evidence for a complex mechanism from super-resolution fluorescence imaging.

Amphiphilic polymers bearing cationic moieties are an emerging alternative to traditional antibiotics given their broad-spectrum activity and low susceptibility to the development of resistance. To date, however, much remains unclear regarding their mechanism of action. Using functional assays (ATP leakage, cell viability, DNA binding) and super-high resolution structured illumination microscopy (OMX-SR) of fluorescently tagged polymers, we present evidence for a complex mechanism, involving membrane permeation as well as cellular uptake, interaction with intracellular targets and possible complexation with bacterial DNA. STATEMENT OF SIGNIFICANCE: This manuscript details the first study to systematically and directly investigate the mechanism of action of antimicrobial polymers, using super-resolution fluorescence imaging as well as functional assays. While many in the field cite membrane permeation as the sole mechanism underlying the activity of such polymers, we present evidence for multimodal actions including high cellular uptake and interaction with intracellular targets. It is also the first report to show competitive binding of antimicrobial polymers with bacterial DNA in a dose-dependent manner.

Selective and Rapid Light-Induced RAFT Single Unit Monomer Insertion in Aqueous Solution.

The photocatalyst Zn(II) meso-tetra(4-sulfonatophenyl)porphyrin (ZnTPPS) is found to substantially accelerate visible-light-initiated (red, yellow, green light) single unit monomer insertion (SUMI) of N,N-dimethylacrylamide into the reversible addition-fragmentation chain transfer (RAFT) agent, 4-((((2-carboxyethyl)thio)carbonothioyl)thio)-4-cyanopentanoic acid (RAFT1 ), in aqueous solution. Thus, under irradiation with red (633 nm) or yellow (593 nm) light with 50 mpm (moles per million mole of monomer) ZnTPPS at 30 °C, the rate enhancement provided by photoinduced energy or electron transfer (PET) is ≈sevenfold over the rate of direct photoRAFT-SUMI (without catalyst), which corresponds to achieving full and selective reaction in hours versus days. Importantly, the selectivity, as judged by the absence of oligomers, is retained. Under green light at similar power, higher rates of SUMI are also observed. However, the degree of enhancement provided by PET-RAFT-SUMI over direct photoRAFT-SUMI as a function of catalyst concentration is less and some oligomers are formed.

Electrochemical Behavior of Thiocarbonylthio Chain Transfer Agents for RAFT Polymerization.

Electrochemical activation of thiocarbonylthio reversible addition-fragmentation chain transfer (RAFT) agents (S=C(Z)S-R) is explored as a potential method for initiating RAFT polymerization under mild conditions without producing initiator-derived byproducts. Herein we apply cyclic voltammetry to establish a predominant reduction mechanism, where electrochemical reduction is coupled to an irreversible first-order chemical reaction. Structure-dependent trends in cyclic voltammograms (CVs), and comparison to absorption spectra, clarify the role of R- and Z-groups in determining reduction processes. The major reduction peak moves to more cathodic potentials in the series dithiobenzoates > trithiocarbonates > heteroaromatic dithiocarbamates > xanthates ∼ N-alkyl-N-aryldithiocarbamates, due to the Z-group influence on thiocarbonyl bond reactivity. More active (electron-withdrawing, radical stabilizing) R-groups shift the reduction peak anodically, in part due to their influence on the rate of the coupled chemical reaction. Analysis of CVs across a range of scan rates revealed that kinetic control over the reduction mechanism is influenced by both the charge transfer rate and chemical reaction rate.

Engineering the Biointerface of Electrospun 3D Scaffolds with Functionalized Polymer Brushes for Enhanced Cell Binding.

Electrospun ultrafine fibers prepared using a blend of poly(lactide- co-glycolide) (PLGA) and bromine terminated poly(l-lactide) (PLA-Br), were surface modified using surface-initiated (SI) Cu(0) mediated polymerization. Copolymers based on N-acryloxysuccinimide (NAS) and a low fouling monomer (either N, N-dimethylacrylamide (DMA), N-(2-hydroxypropyl)acrylamide (HPA), or N-acryloylmorpholine (NAM)) were grafted from the fiber surface to impart surface functionality and to reduce nonspecific protein adsorption. Inclusion of the functional NAS monomer facilitated the conjugation of a nonbioactive cyclic RAD peptide and a bioactive cyclic RGD peptide, the latter expected to facilitate cell adhesion through its affinity for the αvß3 integrin receptor. A detailed analysis of the surface of the electrospun fiber scaffolds in nongrafted form compared to the surface functionalized state is presented. Characteristic amino acid peaks are observed for both conjugated RGD and RAD peptides. Cell culture experiments confirmed cell specific attachment mediated through the presence of the bioactive RGD peptide mainly at high surface density.

Light-Induced RAFT Single Unit Monomer Insertion in Aqueous Solution-Toward Sequence-Controlled Polymers.

First report on the sequential, visible light-initiated, single unit monomer insertion (SUMI) of N,N-dimethylacrylamide (DMAm) into the reversible addition fragmentation chain transfer (RAFT) agent, 4-((((2-carboxyethyl)thio)carbonothioyl)thio)-4-cyanopentanoic acid (CTA1 ), in aqueous solution is provided. The specificity for SUMI over formation of higher oligomers and/or RAFT agent-derived by-products is higher for longer irradiation wavelengths. Red light provides the cleanest product (selective SUMI), showing a linear pseudo-first order kinetic profile to high (>80%) conversion, but also the slowest reaction rate. Blue light provides a relatively rapid reaction, but also gives some by-products (<2%) and the kinetic profile displays a conversion plateau at >65% conversion. Higher specificity with red light is attributed to CTA1 absorbing at longer wavelengths than the SUMI product, which allows selective excitation of CTA1 . The use of a higher reaction temperature (65 °C vs ambient) results in a higher reaction rate and a reduction in oligomer formation.

Optimisation of grafting of low fouling polymers from three-dimensional scaffolds via surface-initiated Cu(0) mediated polymerisation.

Electrospun fibres represent a realistic implantable scaffold containing most of the structural three-dimensional (3D) characteristics of the extracellular matrix. However, as a result of their often synthetic nature, surface energy and chemistry, these scaffolds may adsorb a layer of non-specific proteins which can evoke a foreign body response. The precise surface modification of the scaffolds is challenging due to the complex geometrical and structural organization of the fibre meshes, that may limit the efficacy and completeness of approaches used. One flexible strategy that has gained attention is the use of reversible deactivation radical polymerisation (RDRP) techniques, which allow the creation of polymer brushes with controlled molecular weight, whilst retaining fibre morphology. In this study, protein adsorption was reduced with grafting of poly(N,N-dimethylacrylamide) (PDMA), poly(N-(2-hydroxypropyl)acrylamide) (PHPA) and poly(N-acryloylmorpholine) (PNAM) via surface-initiated (SI)-Cu(0) mediated radical polymerisation, from the surface of electrospun fibres prepared using a blend of bromine terminated poly(l-lactide) (PLA-Br) and poly(d,l-lactide-co-glycolide) (PLGA). Optimisation of the levels of Cu(i)Br, Me6TREN and the presence and concentration of a sacrificial initiator facilitated the grafting of well-controlled polymers brushes in less than one hour. Surface characterisation of the grafted scaffolds using X-ray photoelectron spectroscopy (XPS) and time of flight secondary ion mass spectroscopy (ToF-SIMS), and direct analysis of the molecular weight and polydispersity of polymer formed in solution during the reaction as well as the grafted polymer layer confirmed successful, controlled modification. Finally, protein adsorption experiments demonstrated the low adsorption properties of all polymer coatings with PDMA showing superior performance.

Surface modification of electrospun fibres for biomedical applications: A focus on radical polymerization methods.

The development of electrospun ultrafine fibres from biodegradable and biocompatible polymers has created exciting opportunities for biomedical applications. Fibre meshes with high surface area, suitable porosity and stiffness have been produced. Despite desirable structural and topographical properties, for most synthetic and some naturally occurring materials, the nature of the fibre surface chemistry has inhibited development. Hydrophobicity, undesirable non-specific protein adsorption and bacterial attachment and growth, coupled with a lack of surface functionality in many cases and an incomplete understanding of the myriad of interactions between cells and extracellular matrix (ECM) proteins have impeded the application of these systems. Chemical and physical treatments have been applied in order to modify or control the surface properties of electrospun fibres, with some success. Chemical modification using controlled radical polymerization, referred to here as reversible-deactivation radical polymerization (RDRP), has successfully introduced advanced surface functionality in some fibre systems. Atom transfer radical polymerization (ATRP) and reversible addition fragmentation chain transfer (RAFT) are the most widely investigated techniques. This review analyses the practical applications of electrospinning for the fabrication of high quality ultrafine fibres and evaluates the techniques available for the surface modification of electrospun ultrafine fibres and includes a detailed focus on RDRP approaches.

Triple Activity of Lamivudine Releasing Sulfonated Polymers against HIV-1.

In this article a library of polymeric therapeutic agents against the human immunodeficiency virus (HIV) is presented. The library of statistical copolymers of varied molar mass was synthesized by reversible addition-fragmentation chain transfer (RAFT) polymerization. The synthesized polymers comprise pendent hydroxyl and sulfonated side chains as well as the reverse transcriptase prodrug lamivudine (3TC) attached via a disulfide self-immolative linker. The glutathione mediated release of 3TC is demonstrated as well as the antiviral efficacy against HIV entry and polymerase activity. Although a high degree of polymer sulfonation is required for effective HIV entry inhibition, polymers with approximately ∼50% sulfonated monomer demonstrated potent kinase independent reverse transcriptase inhibition. In addition, the sulfonated polymers demonstrate activity against DNA-DNA polymerase, which suggests that these polymers may exhibit activity against a broad spectrum of viruses. In summary, the polymers described provide a triple-active arsenal against HIV with extracellular activity via entry inhibition and intracellular activity by kinase-dependent lamivudine-based and kinase-independent sulfonated polymer based inhibition. Since these sulfonated copolymers are easily formulated into gels, we envision them to be particularly suited for topical application to prevent the mucosal transmission of viruses, particularly HIV.

Macromolecular prodrugs of ribavirin: towards a treatment for co-infection with HIV and HCV.

Human immunodeficiency virus (HIV) and hepatitis C virus (HCV) represent tremendous healthcare burdens with a large proportion of patients hosting the two viruses at the same time. An altered hepatic function and immunity as well as cross-interference of drugs make treatment of co-infection increasingly challenging. Herein we report the first design of macromolecular prodrugs (MP) with concurrent success in fighting HIV and alleviating hepatitis (liver inflammation). To achieve this, polymer compositions were systematically screened in a broad range of molar mass and content of ribavirin - a broad spectrum antiviral agent. For the first time, we report that ribavirin is efficacious in fighting HIV and in the form of MP, the treatment is safe, both in terms of lack of association of ribavirin with red blood cells and lack of toxicity upon cellular internalization. The lead polymer compositions were also potent in anti-inflammatory assays with relevance to viral hepatitis - thus making up formulations with potential for treatment of co-infection with HIV and HCV.

Enhancement of MHC-I antigen presentation via architectural control of pH-responsive, endosomolytic polymer nanoparticles.

Protein-based vaccines offer a number of important advantages over organism-based vaccines but generally elicit poor CD8(+) T cell responses. We have previously demonstrated that pH-responsive, endosomolytic polymers can enhance protein antigen delivery to major histocompatibility complex class I (MHC-I) antigen presentation pathways thereby augmenting CD8(+) T cell responses following immunization. Here, we describe a new family of nanocarriers for protein antigen delivery assembled using architecturally distinct pH-responsive polymers. Reversible addition-fragmentation chain transfer (RAFT) polymerization was used to synthesize linear, hyperbranched, and core-crosslinked copolymers of 2-(N,N-diethylamino)ethyl methacrylate (DEAEMA) and butyl methacrylate (BMA) that were subsequently chain extended with a hydrophilic N,N-dimethylacrylamide (DMA) segment copolymerized with thiol-reactive pyridyl disulfide (PDS) groups. In aqueous solution, polymer chains assembled into 25 nm micellar nanoparticles and enabled efficient and reducible conjugation of a thiolated protein antigen, ovalbumin. Polymers demonstrated pH-dependent membrane-destabilizing activity in an erythrocyte lysis assay, with the hyperbranched and cross-linked polymer architectures exhibiting significantly higher hemolysis at pH ≤ 7.0 than the linear diblock. Antigen delivery with the hyperbranched and cross-linked polymer architecture enhanced in vitro MHC-I antigen presentation relative to free antigen, whereas the linear construct did not have a discernible effect. The hyperbranched system elicited a four- to fivefold increase in MHC-I presentation relative to the cross-linked architecture, demonstrating the superior capacity of the hyperbranched architecture in enhancing MHC-I presentation. This work demonstrates that the architecture of pH-responsive, endosomolytic polymers can have dramatic effects on intracellular antigen delivery, and offers a promising strategy for enhancing CD8(+) T cell responses to protein-based vaccines.

Polymers fight HIV: potent (pro)drugs identified through parallel automated synthesis.

Macromolecular (pro)drugs interfere with the proliferation of HIV through both inhibition of viral cell entry and via intracellular delivery of antiviral drugs. Lead polymer conjugates exhibit longevity of action exceeding that of parent nucleoside analogue drug and are active in primary T cell over at least 72 h.

Liposomal drug deposits in poly(dopamine) coatings: effect of their composition, cell type, uptake pathway considerations, and shear stress.

Implantable devices equipped with coatings which have the ability to carry and deliver active compounds are of great interest. We report the assembly of liposome-containing poly(dopamine) films, and their interaction with adhering cells. The liposome composition is varied by adding lipophilic dopamine-conjugates and charged lipids. The cell mean fluorescence (CMF) of adhering cells due to the internalization of fluorescent cargo is found to be similar for coatings with the lipophilic-dopamine conjugates, while the charge affects the amount and location of the internalized cargo. The uptake mechanism for cargo by myoblasts using chemical inhibitors is found to be dependent on the used type of liposome. The CMF is significantly reduced for endothelial cells adhering to coatings with applied shear stress.

Novel RAFT amphiphilic brush copolymer steric stabilisers for cubosomes: poly(octadecyl acrylate)-block-poly(polyethylene glycol methyl ether acrylate).

Copolymers, particularly Pluronics®, are typically used to sterically stabilise colloidal nanostructured particles composed of a lyotropic liquid crystalline bicontinuous cubic phase (cubosomes). There is a need to design and assess new functionalisable stabilisers for these colloidal drug delivery systems. Six amphiphilic brush copolymers, poly(octadecyl acrylate)-block-poly(polyethylene glycol methyl ether acrylate) (P(ODA)-b-P(PEGA-OMe)), synthesised by reversible addition-fragmentation chain transfer (RAFT), were assessed as novel steric stabilisers for cubosomes. It was found that increasing the density of PEG on the nanostructured particle surface by incorporating a PEG brush design (i.e., brush copolymer), provided comparable and/or increased stabilisation effectiveness compared to a linear PEG structure, Pluronic® F127, which is extensively used for steric stabilisation of cubosomes. Assessment was conducted both prior to and following the removal of the dodecyl trithiocarbonate end-group, by free radical-induced reduction. The reduced (P(ODA)-b-P(PEGA-OMe) copolymers were more effective steric stabilisers for phytantriol and monoolein colloidal particle dispersions than their non-reduced analogues. High throughput characterisation methodologies, including an accelerated stability assay (ASA) and synchrotron small angle X-ray scattering (SAXS), were implemented in this study for the rapid assessment of steric stabiliser effectiveness and lyotropic liquid crystalline phase identification. Phytantriol cubosomes stabilised with P(ODA)-b-P(PEGA-OMe) copolymers exhibited a double diamond cubic phase (Q(2)(D)), whilst monoolein cubosomes exhibited a primitive cubic phase (Q(2)(P)), analogous to those formed using Pluronic® F127.

Macromolecular prodrugs of ribavirin: concerted efforts of the carrier and the drug.

Polymers in tune. Automated parallel polymer synthesis is developed to obtain libraries of macromolecular prodrugs of ribavirin, a broad-spectrum antiviral agent. As many as 10 identified lead polymer conjugates exhibit therapeutic efficacy matching that of the pristine drug and at the same time suppressed the origin of the main side effect of ribavirin.

Antimicrobial Polymethacrylates Synthesized as Mimics of Tryptophan-Rich Cationic Peptides.

This study describes a facile and high yielding route to two series of polymethacrylates inspired by the naturally occurring, tryptophan-rich cationic antimicrobial polymers. Appropriate optimization of indole content within each gave rise to polymers with high potency against Staphylococcus epidermidis (e.g., PGI-3 minimum inhibitory concentration (MIC) = 12 µg/mL) and the methicillin-resistant strain of Staphylococcus aureus (e.g., PGI-3 MIC = 47 µg/mL) with minimal toxicity toward human red blood cells. Future work will be directed toward understanding the cooperative roles that the cationic and indole pendant groups have for the mechanism of these polymers.

Layer-by-layer polymer coating on discrete particles of cubic lyotropic liquid crystalline dispersions (cubosomes).

Cubic phase lyotropic liquid crystalline colloidal dispersions (cubosomes) were surface-modified with seven polyelectrolyte layers using a layer-by-layer (LbL) approach. The first layer consisted of a copolymer synthesized from methacrylic acid and oleoyl methacrylate for enhanced incorporation within the bilayer of the cubic nanostructure. Six additional layers of poly(L-lysine) and poly(methacrylic acid) were then sequentially added, followed by a washing procedure to remove polymer aggregates from the soft matter particles. Polymer buildup was monitored via microelectrophoresis, dynamic light scattering, and small-angle X-ray scattering. Polymer-coated cubosomes were observed with cryo-transmission electron microscopy. A potential application of the modified nanostructured particles presented in this study is to reduce the burst-release effect associated with drug-loaded cubosomes. The effectiveness of this approach was demonstrated through loading and release results from a model hydrophilic small molecule (fluorescein).