Lipid sulfoxide polymers as potential inhalable drug delivery platforms with differential albumin binding affinity.

Inhalable nanomedicines are increasingly being developed to optimise the pharmaceutical treatment of respiratory diseases. Large lipid-based nanosystems at the forefront of the inhalable nanomedicines development pipeline, though, have a number of limitations. The objective of this study was, therefore, to investigate the utility of novel small lipidated sulfoxide polymers based on poly(2-(methylsulfinyl)ethyl acrylate) (PMSEA) as inhalable drug delivery platforms with tuneable membrane permeability imparted by differential albumin binding kinetics. Linear PMSEA (5 kDa) was used as a hydrophilic polymer backbone with excellent anti-fouling and stealth properties compared to poly(ethylene glycol). Terminal lipids comprising single (1C2, 1C12) or double (2C12) chain diglycerides were installed to provide differing affinities for albumin and, by extension, albumin trafficking pathways in the lungs. Albumin binding kinetics, cytotoxicity, lung mucus penetration and cellular uptake and permeability through key cellular barriers in the lungs were examined in vitro. The polymers showed good mucus penetration and no cytotoxicity over 24 h at up to 1 mg ml-1. While 1C2-showed no interaction with albumin, 1C12-PMSEA and 2C12-PMSEA bound albumin with KD values of approximately 76 and 10 µM, respectively. Despite binding to albumin, 2C12-PMSEA showed reduced cell uptake and membrane permeability compared to the smaller polymers and the presence of albumin had little effect on cell uptake and membrane permeability. While PMSEA strongly shielded these lipids from albumin, the data suggest that there is scope to tune the lipid component of these systems to control membrane permeability and cellular interactions in the lungs to tailor drug disposition in the lungs.

Hydrogenated Soy Phosphatidylcholine Liposomes Stimulate Differential Expression of Chemokines And Cytokines by Rat Alveolar Macrophages In Vitro.

Liposomes are being developed as inhalable drug delivery systems, but concerns remain about their impact on the lungs. To better understand the impact of liposomes and their physicochemical properties on alveolar macrophages, the cytokine and chemokine expression profile of rat alveolar Nr8383 macrophages exposed to 0.1 and 1 mg/ml hydrogenated soy phosphatidylcholine (HSPC) liposomes was examined. Expression patterns varied considerably between liposomes in a concentration-dependent manner, with both anti- and pro-inflammatory chemokines/cytokines produced. Uncharged liposomes induce the greatest production of cytokines and chemokines, followed by PEGylated liposomes. The most significant increase in cytokine/chemokine expression was seen for IL-2 (up to 24-fold), IL-4 (up to 5-fold), IL-18 and VEGF (up to 10-fold), while liposome exposure significantly reduced MIP1 expression (5-fold). In summary, we demonstrate that liposome surface properties promote variable patterns of cytokine and chemokine secretion by alveolar macrophages. This suggests that the type of liposome employed may influence the type of immune response generated in the lung and by extension, dictate how inhaled liposomal nanomedicines affect the lungs response to inhaled toxicants and local infections.

A PEGylated liposomal formulation of prochlorperazine that limits brain exposure but retains dynamin II activity: A potential adjuvant therapy for cancer patients receiving chemotherapeutic mAbs.

Anti-cancer monoclonal antibodies often fail to provide therapeutic benefit in receptor-positive patients due to rapid endocytosis of antibody-bound cell surface receptors. High dose co-administration of prochlorperazine (PCZ) inhibits endocytosis and sensitises tumours to mAbs by inhibiting dynamin II but can also introduce neurological side effects. We examined the potential to use PEGylated liposomal formulations of PCZ (LPCZ) to retain the anti-cancer effects of PCZ, but limit brain uptake. Uncharged liposomes showed complete drug encapsulation and pH-dependent drug release, but cationic liposomes showed limited drug encapsulation and lacked pH-dependent drug release. Uncharged LPCZ showed comparable inhibition of EGFR internalisation to free PCZ in KJD cells. After IV administration to rats, LPCZ reduced the plasma clearance and brain uptake of PCZ compared to IV PCZ. The results suggest that LPCZ may offer some benefit over PCZ as an adjunct therapy in cancer patients receiving mAb treatment.

Lipidated brush-PEG polymers as low molecular weight pulmonary drug delivery platforms.

OBJECTIVES: Nanomedicines are being actively developed as inhalable drug delivery systems. However, there is a distinct utility in developing smaller polymeric systems that can bind albumin in the lungs. We therefore examined the pulmonary pharmacokinetic behavior of a series of lipidated brush-PEG (5 kDa) polymers conjugated to 1C2, 1C12 lipid or 2C12 lipids. METHODS: The pulmonary pharmacokinetics, patterns of lung clearance and safety of polymers were examined in rats. Permeability through monolayers of primary human alveolar epithelia, small airway epithelia and lung microvascular endothelium were also investigated, along with lung mucus penetration and cell uptake. RESULTS: Polymers showed similar pulmonary pharmacokinetic behavior and patterns of lung clearance, irrespective of lipid molecular weight and albumin binding capacity, with up to 30% of the dose absorbed from the lungs over 24 h. 1C12-PEG showed the greatest safety in the lungs. Based on its larger size, 2C12-PEG also showed the lowest mucus and cell membrane permeability of the three polymers. While albumin had no significant effect on membrane transport, the cell uptake of C12-conjugated PEGs were increased in alveolar epithelial cells. CONCLUSION: Lipidated brush-PEG polymers composed of 1C12 lipid may provide a useful and novel alternative to large nanomaterials as inhalable drug delivery systems.

Utilization of endogenous albumin trafficking pathways in the lungs has potential to modestly increase the lung interstitial access and absorption of drug delivery systems after inhaled administration.

OBJECTIVES: Drug delivery systems typically show limited access to the lung interstitium and absorption after pulmonary delivery. The aim of this work was to undertake a proof-of-concept investigation into the potential of employing endogenous albumin and albumin absorption mechanisms in the lungs to improve lung interstitial access and absorption of inhaled drug delivery systems that bind albumin. METHODS: The permeability of human albumin (HSA) through monolayers of primary human alveolar epithelia, small airway epithelia, and microvascular endothelium were investigated. The pulmonary pharmacokinetics of bovine serum albumin (BSA) was also investigated in efferent caudal mediastinal lymph duct-cannulated sheep after inhaled aerosol administration. RESULTS: Membrane permeability coefficient values (Papp) of HSA increased in the order alveolar epithelia

Dose-Dependent Effect of Phenothiazines as Dynamin II Inhibitors on the Uptake of PEGylated Liposomes by Endocytic Cells and In Vivo Pharmacokinetics of PEGylated Liposomal Doxorubicin in Rats.

Dynamin II (dynII) plays a significant role in the internalization pathways of endocytic cells, by allowing membrane invaginations to "bud off". An important class of dynII inhibitors that are used clinically are phenothiazines, such as prochlorperazine (PCZ). PCZ is an antipsychotic drug but is also currently in clinical trials at higher concentrations as an adjuvant in cancer patients that increases the efficacy of monoclonal antibodies at high intravenous doses. It is unknown, however, whether high-dose dynII inhibitors have the potential to alter the pharmacokinetics of co-administered chemotherapeutic nanomedicines that are largely cleared via the mononuclear phagocyte system. This work therefore sought to investigate the impact of clinically relevant concentrations of phenothiazines, PCZ and thioridazine, on in vitro liposome endocytosis and in vivo liposome pharmacokinetics after PCZ infusion in rats. The uptake of fluorescently labeled PEGylated liposomes into differentiated and undifferentiated THP-1 and RAW246.7 cells, and primary human peripheral white blood cells, was investigated via flow cytometry after co-incubation with dynII inhibitors. The IV pharmacokinetics of PEGylated liposomes were also investigated in rats after a 20 min infusion with PCZ. Phenothiazines and dyngo4a reduced the uptake of PEGylated liposomes by THP-1 and RAW264.7 cells in a concentration-dependent manner in vitro. However, dynII inhibitors did not alter the mean uptake of liposomes by human peripheral white blood cells, but endocytic white cells from some donors exhibited sensitivity to phenothiazine exposure. When a clinically relevant dose of PCZ was co-administered with PEGylated liposomal doxorubicin (Caelyx/Doxil) in rats, the pharmacokinetics and biodistribution of liposomes were unaltered. These data suggest that while clinically relevant doses of dynII inhibitors can inhibit the uptake of liposomes by endocytic cells in vitro, they are unlikely to significantly affect the pharmacokinetics of long-circulating, co-administered liposomes.

Dose-Dependent Production of Anti-PEG IgM after Intramuscular PEGylated-Hydrogenated Soy Phosphatidylcholine Liposomes, but Not Lipid Nanoparticle Formulations of DNA, Correlates with the Plasma Clearance of PEGylated Liposomal Doxorubicin in Rats.

PEGylated lipid nanoparticle-based Covid-19 vaccines, including Pfizer's BNT162b2 and Moderna's mRNA-1273, have been shown to stimulate variable anti-PEG antibody production in humans. Anti-PEG antibodies have the potential to accelerate the plasma clearance of PEGylated therapeutics, such as PEGylated liposomes and proteins, and compromise their therapeutic efficacy. However, it is not yet clear whether antibody titers produced by PEGylated Covid-19 vaccines significantly affect the clearance of PEGylated therapeutics. This study examined how anti-PEG IgM levels affect the pharmacokinetics of PEGylated liposomal doxorubicin (PLD) and compared the immunogenicity of a lipid nanoparticle formulation of linear DNA (DNA-LNP) to standard PEG-HSPC liposomes. DNA-LNP was prepared using the same composition and approach as Pfizer's BNT162b2, except linear double-stranded DNA was used as the genetic material. PEGylated HSPC-based liposomes were formulated using the lipid rehydration and extrusion method. Nanoparticles were dosed IM to rats at 0.005-0.5 mg lipid/kg body weight 1 week before evaluating the plasma pharmacokinetics of clinically relevant doses of PLD (1 mg/kg, IV) or PEGylated interferon α2a (Pegasys, 5 µg/kg, SC). Plasma PEG IgM was compared between pre- and 1-week post-dose blood samples. The results showed that anti-PEG IgM production increased with increasing PEG-HSPC liposome dose and that IgM significantly correlated with the plasma half-life, clearance, volume of distribution, and area under the curve of a subsequent dose of PLD. The plasma exposure of Pegasys was also significantly reduced after initial delivery of 0.005 mg/ml PEG-HSPC liposome. However, a single 0.05 mg/kg IM dose of DNA-LNP did not significantly elevate PEG IgM and did not alter the IV pharmacokinetics of PLD. These data showed that PEGylated Covid-19 vaccines are less immunogenic compared to standard PEGylated HSPC liposomes and that there is an antibody threshold for accelerating the clearance of PEGylated therapeutics.

Demonstrating a link between diet, gut microbiota and brain: 14C radioactivity identified in the brain following gut microbial fermentation of 14C-radiolabeled tyrosine in a pig model.

Background: There is a need to better understand the relationship between the diet, the gut microbiota and mental health. Metabolites produced when the human gut microbiota metabolize amino acids may enter the bloodstream and have systemic effects. We hypothesize that fermentation of amino acids by a resistant protein-primed gut microbiota could yield potentially toxic metabolites and disturb the availability of neurotransmitter precursors to the brain. However, these mechanisms are challenging to investigate via typical in vitro and clinical methods. Methods: We developed a novel workflow using 14C radiolabeling to investigate complex nutrient-disease relationships. The first three steps of the workflow are reported here. α-Linolenic acid (ALA) was used as a model nutrient to confirm the efficacy of the workflow, and tyrosine (Tyr) was the test nutrient. 14C-Tyr was administered to male weanling pigs fed a high resistant protein diet, which primed the gut microbiota for fermenting protein. The hypotheses were; (1) that expected biodistribution of 14C-ALA would be observed, and (2) that radioactivity from 14C-Tyr, representing Tyr and other amino acids released from resistant protein following gut microbial fermentation, would be bioavailable to the brain. Results: Radioactivity from the 14C-ALA was detected in tissues reflecting normal utilization of this essential fatty acid. Radioactivity from the 14C-Tyr was detected in the brain (0.15% of original dose). Conclusion: Metabolites of gut-fermented protein and specifically amino acid precursors to neurotransmitters such as tyrosine, are potentially able to affect brain function. By extension, resistant proteins in the diet reaching the gut microbiota, also have potential to release metabolites that can potentially affect brain function. The high specificity of detection of 14C radioactivity demonstrates that the proposed workflow can similarly be applied to understand other key diet and health paradigms.

Using Graph-Based Signatures to Guide Rational Antibody Engineering.

Antibodies are essential experimental and diagnostic tools and as biotherapeutics have significantly advanced our ability to treat a range of diseases. With recent innovations in computational tools to guide protein engineering, we can now rationally design better antibodies with improved efficacy, stability, and pharmacokinetics. Here, we describe the use of the mCSM web-based in silico suite, which uses graph-based signatures to rapidly identify the structural and functional consequences of mutations, to guide rational antibody engineering to improve stability, affinity, and specificity.

Liposomes are Poorly Absorbed via Lung Lymph After Inhaled Administration in Sheep.

Enhancing the delivery of therapeutic agents to the lung lymph, including drugs, transfection agents, vaccine antigens and vectors, has the potential to significantly improve the treatment and prevention of a range of lung-related illnesses. One way in which lymphatic delivery can be optimized is via the use of nanomaterial-based carriers, such as liposomes. After inhaled delivery however, there is conflicting information in the literature regarding whether nanomaterials can sufficiently access the lung lymphatics to have a therapeutic benefit, in large part due to a lack of reliable quantitative pharmacokinetic data. The aim of this work was to quantitatively evaluate the pulmonary lymphatic pharmacokinetics of a model nanomaterial-based drug delivery system (HSPC liposomes) in caudal mediastinal lymph duct cannulated sheep after nebulized administration to the lungs. Liposomes were labelled with 3H-phosphatidylcholine to facilitate evaluation of pharmacokinetics and biodistribution in biological samples. While nanomaterials administered to the lungs may access the lymphatics via direct absorption from the airways or after initial uptake by alveolar macrophages, only 0.3 and 0.001% of the 3H-lipid dose was recovered in lung lymph fluid and lymph cell pellets (containing immune cells) respectively over 5 days. This suggests limited lymphatic access of liposomes, despite apparent pulmonary bioavailability of the 3H-lipid being approximately 17%, likely a result of absorption of liberated 3H-lipid after breakdown of the liposome in the presence of lung surfactant. Similarly, biodistribution of 3H in the mediastinal lymph node was insignificant after 5 days. These data suggest that liposomes, that are normally absorbed via the lymphatics after interstitial administration, do not access the lung lymphatics after inhaled administration. Alternate approaches to maximize the lung lymphatic delivery of drugs and other therapeutics need to be identified.

Development of a hyperbranched polymer-based methotrexate nanomedicine for rheumatoid arthritis.

Methotrexate (MTX) is an effective disease modifying anti-rheumatic drug, but can cause significant hepatotoxicity and liver failure in some individuals. The goal of this work was to develop a MTX-conjugated hyperbranched polymeric nanoparticle based on oligo(ethylene glycol) methyl ether methacrylate (OEGMA) and examine its ability to selectively deliver MTX to rheumatic joints while sparing the liver. MTX was conjugated to the hyperbranched polymer via a matrix metalloproteinase-13 cleavable peptide linker. Two populations of nanoparticles were produced, with sizes averaging 20 and 200nm. Tri-peptide (FFK)-modified MTX was liberated in the presence of matrix metalloproteinase 13 (MMP-13)and showed 100 to 1000-fold lower antiproliferative capacity in monocytic THP-1 cells compared to unmodified MTX, depending on whether the gamma-carboxylate of MTX was functionalized with O-tert-butyl. Nanoparticles showed prolonged plasma exposure after intravenous injection with a terminal half-life of approximately 1 day, but incomplete (50%) absorption after subcutaneous administration. Nanoparticles selectively accumulated in inflamed joints in a rat model of rheumatoid arthritis and showed less than 5% biodistribution in the liver after 5 days. MTX-OtBu nanoparticles also showed no hepatocellular toxicity at 500 µM MTX equivalents. This work provides support for the further development of OEGMA-based hyperbranched polymers as MTX drug delivery systems for rheumatoid arthritis. STATEMENT OF SIGNIFICANCE: Nanomedicines containing covalently conjugated methotrexate offer the potential for selective accumulation of the potent hepatotoxic drug in rheumatic joints and limited liver exposure. One limitation of the high surface presentation of methotrexate on a nanoparticle surface, however, is the potential for enhanced liver uptake. We developed several OEGMA-based hyperbranched polymers containing alpha-carboxyl modified and unmodified methotrexate conjugated via an MMP-13 cleavable hexapeptide linker. The modified methotrexate polymer showed promising in vitro and in vivo behavior warranting further development and optimization as an anti-rheumatic nanomedicine. This work presents a new avenue for further research into the development of hyperbranched polymers for rheumatoid arthritis and suggests interesting approaches that may overcome some limitations associated with the translation of anti-rheumatic nanomedicines into patients.

Poly(HPMA-co-NIPAM) copolymer as an alternative to polyethylene glycol-based pharmacokinetic modulation of therapeutic proteins.

PEGylation is the standard approach for prolonging the plasma exposure of protein therapeutics but has limitations. We explored whether polymers prepared by Reversible Addition-Fragmentation chain-Transfer (RAFT) may provide better alternatives to polyethylene glycol (PEG). Four RAFT polymers were synthesised with varying compositions, molar mass (Mn), and structures, including a homopolymer of N-(2-hydroxypropyl)methacrylamide, (pHPMA) and statistical copolymers of HPMA with poly(ethylene glycol methyl ether acrylate) p(HPMA-co-PEGA); HPMA and N-acryloylmorpholine, p(HPMA-co-NAM); and HPMA and N-isopropylacrylamide, p(HPMA-co-NIPAM). The intravenous pharmacokinetics of the polymers were then evaluated in rats. The in vitro activity and in vivo pharmacokinetics of p(HPMA-co-NIPAM)-conjugated trastuzumab Fab' and full length mAb were then evaluated. p(HPMA-co-NIPAM) prolonged plasma exposure more avidly compared to the other p(HPMA) polymers or PEG, irrespective of molecular weight. When conjugated to trastuzumab-Fab', p(HPMA-co-NIPAM) prolonged plasma exposure of the Fab' similar to PEG-Fab'. The generation of anti-PEG IgM in rats 7 days after intravenous and subcutaneous dosing of p(HPMA-co-NIPAM) conjugated trastuzumab mAb was also examined and was shown to exhibit lower immunogenicity than the PEGylated construct. These data suggest that p(HPMA-co-NIPAM) has potential as a promising copolymer for use as an alternative conjugation strategy to PEG, to prolong the plasma exposure of therapeutic proteins.

Nitroxide-functional PEGylated nanostars arrest cellular oxidative stress and exhibit preferential accumulation in co-cultured breast cancer cells.

The limited application of traditional antioxidants to reducing elevated levels of reactive oxygen species (ROS) is potentially due to their lack of stability and biocompatibility when tested in a biological milieu. For instance, the poor biological antioxidant performance of small molecular nitroxides arises from their limited diffusion across cell membranes and their significant side effects when applied at high doses. Herein, we describe the use of nanostructured carriers to improve the antioxidant activity of a typical nitroxide derivative, (2,2,6,6-tetramethylpiperidin-1-yl)oxyl (TEMPO). Polymers with star-shaped structures were synthesised and were further conjugated to TEMPO moieties via amide linkages. The TEMPO-loaded stars have small hydrodynamic sizes (<20 nm), and are better tolerated by cells than free TEMPO in a breast cancer-fibroblast co-culture, a system exhibiting elevated ROS levels. At a well-tolerated concentration, the polymer with the highest TEMPO-loading capacity successfully downregulated ROS production in co-cultured cells (a significant decrease of up to 50% vs. basal ROS levels), which was accompanied by a specific reduction in superoxide anion generation in the mitochondria. In contrast, the equivalent concentration of free TEMPO did not achieve the same outcome. Further investigation showed that the TEMPO-conjugated star polymers can be recycled inside the cells, thus providing longer term scavenging activity. Cell association studies demonstrated that the polymers can be taken up by both cell types in the co-culture, and are found to co-locate with the mitochondria. Interestingly the stars exhibited preferential mitochodria targeting in the co-cultured cancer cells compared to accompanying fibroblasts. The data suggest the potential of TEMPO-conjugated star polymers to arrest oxidative stress for various applications in cancer therapy.

The pharmacokinetics of PEGylated liposomal doxorubicin are not significantly affected by sex in rats or humans, but may be affected by immune dysfunction.

PEGylated liposomal doxorubicin (PLD, Caelyx®, Doxil®) has been suggested to show significant sex-based differences in plasma clearance, as well as high inter-individual variability that may be driven by monocyte counts in cancer patients. This study aimed to establish if these differences are similarly observed in rats, which exhibit similar liposome clearance mechanisms to humans, and to use this model to identify sources of inter-individual and sex-based pharmacokinetic variability. The plasma and lymphatic pharmacokinetics of PLD were evaluated in male and female rats by quantifying doxorubicin as well as the 3H-labelled liposome. In general, the pharmacokinetics of doxorubicin and the 3H-liposome did not differ significantly between male and female rats when corrected for body surface area. Female rats did, however, show significantly higher doxorubicin concentrations in lymph compared to male rats. With the exception of serum testosterone concentrations in males, none of the physiological parameters evaluated correlated with plasma clearance. Further, reanalysis of published human data that formerly reported sex-differences in PLD plasma clearance similarly revealed no significant differences in PLD plasma clearance between males and females with solid tumours, but increased plasma clearance in patients with Kaposi's sarcoma (generally HIV+/immunocompromised). These data suggest that with the exception of lymphatic exposure, there are unlikely to be significant sex effects in the pharmacokinetics of liposomes, but immune function may contribute to inter individual variability.

Recent advances in nano/microparticle-based oral vaccines.

Background: Vaccines are often recognized as one of the most cost-effective public health interventions in controlling infectious diseases. Most pathogens infiltrate the body from mucosal sites, primarily from the oral and pulmonary region and reach the systemic circulation where disease manifestation starts. Traditional needle-based vaccines are usually not capable of inducing immunity at the mucosal sites where pathogen infiltrates start, but induces systemic immunity. In contrast to needle-based vaccines, mucosally administered vaccines induce immunity at both the mucosal sites and systemically. The oral route of immunization is the most convenient way to administer the vaccines. However, due to the complicated and hostile gastrointestinal structure and environment, vaccines need to overcome major hurdles while retaining their stability and immunogenicity. Area covered: This review will briefly discuss different barriers to oral vaccine development. It gives a brief overview of different types of nano/microparticle-based oral vaccines and discusses how physicochemical characteristics of the particles influence overall immunity after oral immunization. Expert opinion: Formulation strategies using novel lipid and polymer-based nano/microparticle platforms retain stability and antigenicity of vaccines against the harsh gastrointestinal condition. The physicochemical properties of particles can be uniquely tailored to prolong the release of antigens, and attached ligands (M-cells and APC-ligands) can precisely target uptake by immune cells. These represent viable strategies for efficient delivery of oral vaccines.

Monocytes Do Not Contribute to Sex Differences Seen in the Pharmacokinetics of Pegylated Liposomal Doxorubicin.

Pegylated liposomal doxorubicin (PLD) is widely utilised in cancer chemotherapy, but exhibits large inter-individual pharmacokinetic variability and sex differences in plasma clearance. Population pharmacokinetic modelling has suggested PLD plasma clearance correlates with peripheral monocyte counts, while sex hormones are known to affect endocytosis and phagocytosis in mononuclear cells. This study investigated whether sex hormones affect the uptake of PLD by human monocytes and macrophages in vitro. 17ß-estradiol, but not testosterone, inhibited the uptake of PLD in a concentration dependent manner in undifferentiated (but not differentiated) THP-1 cells, and primary monocytes obtained from women, but not men. Effects of estradiol were only evident at very high concentrations seen during pregnancy. No differences were observed in monocyte count or monocyte subtypes between males and females. These data show that monocytes do not contribute to sex differences seen in PLD clearance in humans of reproductive age.

Depolymerization of hyaluronan using PEGylated human recombinant hyaluronidase promotes nanoparticle tumor penetration.

Aim: Delivery of nanoparticles (NPs) to tumors can be impeded by high levels of hyaluronan (HA) in the stroma. Enzymatic depolymerization of HA with PEGylated hyaluronidase (PEGPH20) improves the delivery of antibodies to tumors. However, it is unknown whether NP delivery is enhanced by this strategy. Methods: The impact of PEGPH20 pretreatment on the uptake and tumor penetration of model PEGylated polystyrene NPs was studied in mice with orthotopic breast cancers. Results: Tumor oxygenation and NP penetration, but not overall tumor uptake, of 50 nm NPs, was significantly enhanced by PEGPH20 pre-administration. Conclusion: PEGPH20 has the potential to improve intratumoral penetration of NP-based drug delivery systems and enhance access to cancer cells in poorly vascularized regions of the tumor.

Trisulfide linked cholesteryl PEG conjugate attenuates intracellular ROS and collagen-1 production in a breast cancer co-culture model.

The progression of cancer has been closely-linked with augmentation of cellular reactive oxygen species (ROS) levels and ROS-associated changes in the tumour microenvironment (TME), including alterations to the extracellular matrix and associated low drug uptake. Herein we report the application of a co-culture model to simulate the ROS based cell-cell interactions in the TME using fibroblasts and breast cancer cells, and describe how novel reactive polymers can be used to modulate those interactions. Under the co-culture conditions, both cell types exhibited modifications in behaviour, including significant overproduction of ROS in the cancer cells, and elevation of the collagen-1 secretion and stained actin filament intensity in the fibroblasts. To examine the potential of using reactive antioxidant polymers to intercept ROS communication and thereby manipulate the TME, we employed H2S-releasing macromolecular conjugates which have been previously demonstrated to mitigate ROS production in HEK cells. The specific conjugate used, mPEG-SSS-cholesteryl (T), significantly reduced ROS levels in co-cultured cancer cells by approximately 50%. This reduction was significantly greater than that observed with the other positive antioxidant controls. Exposure to T was also found to downregulate levels of collagen-1 in the co-cultured fibroblasts, while exhibiting less impact on cells in mono-culture. This would suggest a possible downstream effect of ROS-mitigation by T on stromal-tumour cell signalling. Since fibroblast-derived collagens modulate crucial steps in tumorigenesis, this ROS-associated effect could potentially be harnessed to slow cancer progression. The model may also be beneficial for interrogating the impact of antioxidants on naturally enhanced ROS levels, rather than relying on the application of exogenous oxidants to simulate elevated ROS levels.

The Impact of Polymer Size and Cleavability on the Intravenous Pharmacokinetics of PEG-Based Hyperbranched Polymers in Rats.

A better understanding of the impact of molecular size and linkers is important for PEG-based hyperbranched polymers (HBPs) intended as tailored drug delivery vehicles. This study aimed to evaluate the effects of crosslinker chemistry (cleavable disulphide versus non-cleavable ethylene glycol methacrylate (EGDMA) linkers) and molecular weight within the expected size range for efficient renal elimination (22 vs. 48 kDa) on the intravenous pharmacokinetic and biodistribution properties of 89Zr-labelled HBPs in rats. All HBPs showed similar plasma pharmacokinetics over 72 h, despite differences in linker chemistry and size. A larger proportion of HBP with the cleavable linker was eliminated via the urine and faeces compared to a similar-sized HBP with the non-cleavable linker, while size had no impact on the proportion of the dose excreted. The higher molecular weight HBPs accumulated in organs of the mononuclear phagocyte system (liver and spleen) more avidly than the smaller HBP. These results suggest that HBPs within the 22 to 48 kDa size range show no differences in plasma pharmacokinetics, but distinct patterns of organ biodistribution and elimination are evident.

Trisulfide-Bearing PEG Brush Polymers Donate Hydrogen Sulfide and Ameliorate Cellular Oxidative Stress.

A potential approach to combat cellular dysfunction is to manipulate cell communication and signaling pathways to restore physiological functions while protecting unaffected cells. For instance, delivering the signaling molecule H2S to certain cells has been shown to restore cell viability and re-normalize cell behavior. We have previously demonstrated the ability to incorporate a trisulfide-based H2S-donating moiety into linear polymers with good in vitro releasing profiles and demonstrated their potential for ameliorating oxidative stress. Herein, we report two novel series of brush polymers decorated with higher numbers of H2S-releasing segments. These materials contain two trisulfide-based monomers co-polymerized with oligo(ethylene glycol methyl ether methacrylate) via reversible addition-fragmentation chain-transfer polymerization. The macromolecules were characterized to have a range of trisulfide densities with similar, well-defined molecular weight distribution, good H2S-releasing profiles, and high cellular tolerance. Using an amperometric technique, the H2S liberated and total sulfide release were found to depend on concentrations and chemical nature of triggering molecules (glutathione and cysteine) and, importantly, the position of reactive groups within the brush structure. Notably, when introduced to cells at well-tolerated doses, two macromolecular donors which have the same proportion as of the H2S-donating monomer (30%) but differ in releasing moiety location show similar cellular H2S-releasing kinetics. These donors can restore reactive oxygen species levels to baseline values, when polymer pretreated cells are exposed to exogenous oxidants (H2O2). Our work opens up a new aspect in preparing H2S macromolecule donors and their application to arresting cellular oxidative cascades.