Versatile Click Linker Enabling Native Peptide Release from Nanocarriers upon Redox Trigger.

Nanocarriers have shown their ability to extend the circulation time of drugs, enhance tumor uptake, and tune drug release. Therapeutic peptides are a class of drug compounds in which nanocarrier-mediated delivery can potentially improve their therapeutic index. To this end, there is an urgent need for orthogonal covalent linker chemistry facilitating the straightforward on-the-resin peptide generation, nanocarrier conjugation, as well as the triggered release of the peptide in its native state. Here, we present a copper-free clickable ring-strained alkyne linker conjugated to the N-terminus of oncolytic peptide LTX-315 via standard solid-phase peptide synthesis (SPPS). The linker contains (1) a recently developed seven-membered ring-strained alkyne, 3,3,6,6-tetramethylthiacycloheptyne sulfoximine (TMTHSI), (2) a disulfide bond, which is sensitive to the reducing cytosolic and tumor environment, and (3) a thiobenzyl carbamate spacer enabling release of the native peptide upon cleavage of the disulfide via 1,6-elimination. We demonstrate convenient "clicking" of the hydrophilic linker-peptide conjugate to preformed pegylated core-cross-linked polymeric micelles (CCPMs) of 50 nm containing azides in the hydrophobic core under aqueous conditions at room temperature resulting in a loading capacity of 8 mass % of peptide to polymer (56% loading efficiency). This entrapment of hydrophilic cargo into/to a cross-linked hydrophobic core is a new and counterintuitive approach for this class of nanocarriers. The release of LTX-315 from the CCPMs was investigated in vitro and rapid release upon exposure to glutathione (within minutes) followed by slower 1,6-elimination (within an hour) resulted in the formation of the native peptide. Finally, cytotoxicity of LTX CCPMs as well as uptake of sulfocyanine 5-loaded CCPMs was investigated by cell culture, demonstrating successful tumor cell killing at concentrations similar to that of the free peptide treatment.

Tunable polymeric micelles for taxane and corticosteroid co-delivery.

Nanomedicine holds promise for potentiating drug combination therapies. Increasing (pre)clinical evidence is available exemplifying the value of co-formulating and co-delivering different drugs in modular nanocarriers. Taxanes like paclitaxel (PTX) are widely used anticancer agents, and commonly combined with corticosteroids like dexamethasone (DEX), which besides for suppressing inflammation and infusion reactions, are increasingly explored for modulating the tumor microenvironment towards enhanced nano-chemotherapy delivery and efficacy. We here set out to develop a size- and release rate-tunable polymeric micelle platform for co-delivery of taxanes and corticosteroids. We synthesized amphiphilic mPEG-b-p(HPMAm-Bz) block copolymers of various molecular weights and used them to prepare PTX and DEX single- and double-loaded micelles of different sizes. Both drugs could be efficiently co-encapsulated, and systematic comparison between single- and co-loaded formulations demonstrated comparable physicochemical properties, encapsulation efficiencies, and release profiles. Larger micelles showed slower drug release, and DEX release was always faster than PTX. The versatility of the platform was exemplified by co-encapsulating two additional taxane-corticosteroid combinations, demonstrating that drug hydrophobicity and molecular weight are key properties that strongly contribute to drug retention in micelles. Altogether, our work shows that mPEG-b-p(HPMAm-Bz) polymeric micelles serve as a tunable and versatile nanoparticle platform for controlled co-delivery of taxanes and corticosteroids, thereby paving the way for using these micelles as a modular carrier for multidrug nanomedicine.

Design of experiments approach for the development of a validated method to determine the exenatide content in poly(lactide-co-glycolide) microspheres.

Due to the lack of pharmacopeia guidelines for injectable microspheres based on poly (D, L-lactide-co-glycolide) (PLGA), an internal method validation is a critical prerequisite for quality assurance. One of the essential issues of developing peptide-based drugs loaded PLGA microspheres is the precise determination of the amount of peptide drug entrapped in the microspheres. The aim of this study is the development and optimization of a method for measuring the drug content loading of PLGA microspheres using exenatide as a model peptide drug. Exenatide-loaded PLGA microspheres were prepared by a double emulsion solvent evaporation method. The extraction method to determine exenatide content in microspheres was optimized using Design of Experiments (DoE) approach. After the initial screening of six factors, using Fractional Factorial design (FFD), four of them, including type of organic solvent, buffer/organic solvent ratio (v/v), shaking time and pH, exhibited significant effects on the response, namely the exenatide loading, and a Box-Behnken design (BBD) was subsequently applied to obtain its optimum level. The optimum level for organic solvent volume, buffer/organic solvent ratio, shaking time, and pH were 4 ml, 1, 5.6 hrs, and pH 6, respectively. The exenatide content in microspheres under these conditions was 6.4 ± 0.0 (%w/w), whereas a value of 6.1% was predicted by the derived equation. This excellent agreement between the actual and the predicted value demonstrates that the fitted model can thus be used to determine the exenatide content.

Effect of Polyplex Size on Penetration into Tumor Spheroids.

Ovarian cancer is one of the most lethal gynecological cancers in the world. In recent years, nucleic acid (NA)-based formulations have been shown to be promising treatments for ovarian cancer, including tumor nodules. However, gene therapy is not that far advanced in clinical reality due to unfavorable physicochemical properties of the NAs, such as high molecular weight, poor cellular uptake, rapid degradation by nucleases, etc. One of the strategies used to overcome these drawbacks is the complexation of anionic NAs via electrostatic interactions with cationic polymers, resulting in the formation of so-called polyplexes. In this work, the role of the size of pDNA and siRNA polyplexes on their penetration into ovarian-cancer-based tumor spheroids was investigated. For this, a methoxypoly(ethylene glycol) poly(2-(dimethylamino)ethyl methacrylate) (mPEG-pDMAEMA) diblock copolymer was synthesized as a polymeric carrier for NA binding and condensation with either plasmid DNA (pDNA) or short interfering RNA (siRNA). When prepared in HEPES buffer (10 mM, pH 7.4) at a nitrogen/phosphate (N/P) charge ratio of 5 and pDNA polyplexes were formed with a size of 162 ± 11 nm, while siRNA-based polyplexes displayed a size of 25 ± 2 nm. The polyplexes had a slightly positive zeta potential of +7-8 mV in the same buffer. SiRNA and pDNA polyplexes were tracked in vitro into tumor spheroids, resembling in vivo avascular ovarian tumor nodules. For this purpose, reproducible spheroids were obtained by coculturing ovarian carcinoma cells with primary mouse embryonic fibroblasts in different ratios (5:2, 1:1, and 2:5). Penetration studies revealed that after 24 h of incubation, siRNA polyplexes were able to penetrate deeper into the homospheroids (composed of only cancer cells) and heterospheroids (cancer cells cocultured with fibroblasts) compared to pDNA polyplexes which were mainly located in the rim. The penetration of the polyplexes was slowed when increasing the fraction of fibroblasts present in the spheroids. Furthermore, in the presence of serum siRNA polyplexes encoding for luciferase showed a high cellular uptake in 2D cells resulting in ∼50% silencing of luciferase expression. Taken together, these findings show that self-assembled small siRNA polyplexes have good potential as a platform to test ovarian tumor nodulus penetration..

Mechanistic Study on the Degradation of Hydrolysable Core-Crosslinked Polymeric Micelles.

Core-crosslinked polymeric micelles (CCPMs) are an attractive class of nanocarriers for drug delivery. Two crosslinking approaches to form CCPMs exist: either via a low-molecular-weight crosslinking agent to connect homogeneous polymer chains with reactive handles or via cross-reactive handles on polymers to link them to each other (complementary polymers). Previously, CCPMs based on methoxy poly(ethylene glycol)-b-poly[N-(2-hydroxypropyl) methacrylamide-lactate] (mPEG-b-PHPMAmLacn) modified with thioesters were crosslinked via native chemical ligation (NCL, a reaction between a cysteine residue and thioester resulting in an amide bond) using a bifunctional cysteine containing crosslinker. These CCPMs are degradable under physiological conditions due to hydrolysis of the ester groups present in the crosslinks. The rapid onset of degradation observed previously, as measured by the light scattering intensity, questions the effectiveness of crosslinking via a bifunctional agent. Particularly due to the possibility of intrachain crosslinks that can occur using such a small crosslinker, we investigated the degradation mechanism of CCPMs generated via both approaches using various analytical techniques. CCPMs based on complementary polymers degraded slower at pH 7.4 and 37 °C than CCPMs with a crosslinker (the half-life of the light scattering intensity was approximately 170 h versus 80 h, respectively). Through comparative analysis of the degradation profiles of the two different CCPMs, we conclude that partially ineffective intrachain crosslinks are likely formed using the small crosslinker, which contributed to more rapid CCPM degradation. Overall, this study shows that the type of crosslinking approach can significantly affect degradation kinetics, and this should be taken into consideration when developing new degradable CCPM platforms.

Lipid nanoparticle-mediated messenger RNA delivery for ex vivo engineering of natural killer cells.

Natural killer (NK) cells participate in the immune system by eliminating cancer and virally infected cells through germline-encoded surface receptors. Their independence from prior activation as well as their significantly lower toxicity have placed them in the spotlight as an alternative to T cells for adoptive cell therapy (ACT). Engineering NK cells with mRNA has shown great potential in ACT by enhancing their tumor targeting and cytotoxicity. However, mRNA transfection of NK cells is challenging, as the most common delivery methods, such as electroporation, show limitations. Therefore, an alternative non-viral delivery system that enables high mRNA transfection efficiency with preservation of the cell viability would be beneficial for the development of NK cell therapies. In this study, we investigated both polymeric and lipid nanoparticle (LNP) formulations for eGFP-mRNA delivery to NK cells, based on a dimethylethanolamine and diethylethanolamine polymeric library and on different ionizable lipids, respectively. The mRNA nanoparticles based on cationic polymers showed limited internalization by NK cells and low transfection efficiency. On the other hand, mRNA-LNP formulations were optimized by tailoring the lipid composition and the microfluidic parameters, resulting in a high transfection efficiency (∼100%) and high protein expression in NK cells. In conclusion, compared to polyplexes and electroporation, the optimized LNPs show a greater transfection efficiency and higher overall eGFP expression, when tested in NK (KHYG-1) and T (Jurkat) cell lines, and cord blood-derived NK cells. Thus, LNP-based mRNA delivery represents a promising strategy to further develop novel NK cell therapies.

Self-Healing Thermosensitive Hydrogel for Sustained Release of Dexamethasone for Ocular Therapy.

The aim of this study was to develop an injectable hydrogel delivery system for sustained ocular delivery of dexamethasone. To this end, a self-healing hydrogel consisting of a thermosensitive ABA triblock copolymer was designed. The drug was covalently linked to the polymer by copolymerization of methacrylated dexamethasone with N-isopropylacrylamide (NIPAM) and N-acryloxysuccinimide (NAS) through reversible addition-fragmentation chain transfer (RAFT) polymerization, using poly(ethylene glycol) (PEG) functionalized at both ends with a chain transfer agent (CTA). Hydrogel formation was achieved by mixing aqueous solutions of the formed thermosensitive polymer (with a cloud point of 23 °C) with cystamine at 37 °C, to result in covalent cross-linking due to the reaction of the N-hydroxysuccimide (NHS) functionality of the polymer and the primary amines of cystamine. Rheological analysis showed both thermogelation and covalent cross-linking at 37 °C, as well as the self-healing properties of the formed network, which was attributed to the presence of disulfide bonds in the cystamine cross-links, making the system injectable. The release of dexamethasone from the hydrogel occurred through ester hydrolysis following first-order kinetics in an aqueous medium at pH 7.4 over 430 days at 37 °C. Based on simulations, administration of 100 mg of hydrogel would be sufficient for maintaining therapeutic levels of dexamethasone in the vitreous for at least 500 days. Importantly, dexamethasone was released from the hydrogel in its native form as determined by LC-MS analysis. Cytocompatibility studies showed that at clinically relevant concentrations, both the polymer and the cross-linker were well tolerated by adult retinal pigment epithelium (ARPE-19) cells. Moreover, the hydrogel did not show any toxicity to ARPE-19 cells. The injectability of the hydrogel, together with the long-lasting release of dexamethasone and good cytocompatibility with a retinal cell line, makes this delivery system an attractive candidate for treatment of ocular inflammatory diseases.

Evaluation of paclitaxel-loaded polymeric nanoparticles in 3D tumor model: impact of tumor stroma on penetration and efficacy.

Since tumor stroma poses as a barrier to achieve efficacy of nanomedicines, it is essential to evaluate nano-chemotherapeutics in stroma-mimicking 3D models that reliably predict their behavior regarding these hurdles limiting efficacy. In this study, we evaluated the effect of paclitaxel-loaded polymeric micelles (PTX-PMCs) and polymeric nanoparticles (PTX-PNPs) in a tumor stroma-mimicking 3D in vitro model. PTX-PMCs (77 nm) based on a amphiphilic block copolymer of mPEG-b-p(HPMAm-Bz) and PTX-PNPs (159 nm) based on poly(lactic-co-glycolic acid) were prepared, which had an encapsulation efficiency (EE%) of 81 ± 15% and 45 ± 8%, respectively. 3D homospheroids of mouse 4T1 breast cancer cells and heterospheroids of NIH3T3 fibroblasts and 4T1 (5:1 ratio) were prepared and characterized with high content two-photon microscopy and immunostaining. Data showed an induction of epithelial-mesenchymal transition (α-SMA) in both homo- and heterospheroids, while ECM (collagen) deposition only in heterospheroids. Two-photon imaging revealed that both fluorescently labeled PMCs and PNPs penetrated into the core of homospheroids and only PMCs penetrated into heterospheroids. Furthermore, PTX-PMCs, PTX-PNPs, and free PTX induced cytotoxicity in tumor cells and fibroblasts grown as monolayer, but these effects were substantially reduced in 3D models, in particular in heterospheroids. Gene expression analysis showed that heterospheroids had a significant increase of drug resistance markers (Bcl2, Abgc2) compared to 2D or 3D monocultures. Altogether, this study shows that the efficacy of nanotherapeutics is challenged by stroma-induced poor penetration and development of resistant phenotype. Therefore, this tumor stroma-mimicking 3D model can provide an excellent platform to study penetration and effects of nanotherapeutics before in vivo studies.

Orthogonal Covalent Entrapment of Cargo into Biodegradable Polymeric Micelles via Native Chemical Ligation.

Polymeric micelles (PMs) are promising platforms for enhanced tissue targeting of entrapped therapeutic agents. Strategies to circumvent premature release of entrapped drugs include cross-linking of the micellar core as well as covalent attachment of the drug cargo. The chemistry employed to obtain cross-linked micelles needs to be mild to also allow entrapment of fragile molecules, such as certain peptides, proteins, oligonucleotides, and fluorescent dyes. Native chemical ligation (NCL) is a mild bio-orthogonal reaction between a N-terminal cysteine residue and a thioester that proceeds under physiological conditions. Here, we designed a trifunctional cross-linker containing two cysteine residues for the micelle core-cross-linking reaction and an azide residue for ring-strained alkyne conjugation of fluorescent dyes. We applied this approach to thermosensitive methoxypolyethylene glycol-b-N-(2-hydroxypropyl)methacrylamide-lactate (mPEG-b-HPMAmLacn) based block copolymers of a core-cross-linked polymeric micelle (CCPM) system by attaching thioester residues (using ethyl thioglycolate-succinic anhydride, ETSA) for NCL cross-linking with the trifunctional cross-linker under physiological conditions. By use of mild copper-free click chemistry, we coupled fluorescent dyes, Sulfo.Cy5 and BODIPY, to the core via the azide residue present on the cross-linker by triazole ring formation. In addition, we employed a recently developed cycloheptyne strain promoted click reagent (TMTHSI, CliCr) in comparison to the frequently employed cyclooctyne derivative (DBCO), both achieving successful dye entrapment. The size of the resulting CCPMs could be tuned between 50 and 100 nm by varying the molecular weight of the thermosensitive block and ETSA content. In vitro cell experiments showed successful internalization of the dye entrapped CCPMs, which did not affect cell viability up to a polymer concentration of 2 mg/mL in PC3 cells. These fluorescent dye entrapped CCPMs can be applied in diagnostic imaging and the chemistry developed in this study serves as a steppingstone toward covalently entrapped fragile drug compounds with tunable release in CCPMs.

The Impact of Nanobody Density on the Targeting Efficiency of PEGylated Liposomes.

Nanoparticles (NPs) are commonly modified with tumor-targeting moieties that recognize proteins overexpressed on the extracellular membrane to increase their specific interaction with target cells. Nanobodies (Nbs), the variable domain of heavy chain-only antibodies, are a robust targeting ligand due to their small size, superior stability, and strong binding affinity. For the clinical translation of targeted Nb-NPs, it is essential to understand how the number of Nbs per NP impacts the receptor recognition on cells. To study this, Nbs targeting the hepatocyte growth factor receptor (MET-Nbs) were conjugated to PEGylated liposomes at a density from 20 to 800 per liposome and their targeting efficiency was evaluated in vitro. MET-targeted liposomes (MET-TLs) associated more profoundly with MET-expressing cells than non-targeted liposomes (NTLs). MET-TLs with approximately 150-300 Nbs per liposome exhibited the highest association and specificity towards MET-expressing cells and retained their targeting capacity when pre-incubated with proteins from different sources. Furthermore, a MET-Nb density above 300 Nbs per liposome increased the interaction of MET-TLs with phagocytic cells by 2-fold in ex vivo human blood compared to NTLs. Overall, this study demonstrates that adjusting the MET-Nb density can increase the specificity of NPs towards their intended cellular target and reduce NP interaction with phagocytic cells.

Design, development and clinical translation of CriPec®-based core-crosslinked polymeric micelles.

Nanomedicines are used to improve the efficacy and safety of pharmacotherapeutic interventions. Unraveling the biological behavior of nanomedicines, including their biodistribution and target site accumulation, is essential to establish design criteria that contribute to superior performance. CriPec® technology is based on amphiphilic methoxy-poly(ethylene glycol)-b-poly[N-(2-hydroxypropyl) methacrylamide lactate] (mPEG-b-pHPMAmLacn) block copolymers, which are designed to upon self-assembly covalently entrap active pharmaceutical ingredients (API) in core-crosslinked polymeric micelles (CCPM). Key features of CCPM are a prolonged circulation time, high concentrations at pathological sites, and low levels of accumulation in the majority of healthy tissues. Proprietary hydrolysable linkers allow for tunable and sustained release of entrapped API, including hydrophobic and hydrophilic small molecules, as well as peptides and oligonucleotides. Preclinical imaging experiments provided valuable information on their tumor and tissue accumulation and distribution, as well as on uptake by cancer, healthy and immune cells. The frontrunner formulation CPC634, which refers to 65 nm-sized CCPM entrapping the chemotherapeutic drug docetaxel, showed excellent pharmacokinetic properties, safety, tumor accumulation and antitumor efficacy in multiple animal models. In the clinic, CPC634 also demonstrated favorable pharmacokinetics, good tolerability, signs of efficacy, and enhanced localization in tumor tissue as compared to conventional docetaxel. PET imaging of radiolabeled CPC634 showed quantifiable accumulation in âˆ¼50 % of tumors and metastases in advanced-stage cancer patients, and demonstrated potential for use in a theranostic setting even when applied at a companion diagnostic dose. Altogether, the preclinical and clinical results obtained to date demonstrate that mPEG-b-pHPMAmLacn CCPM based on CriPec® technology are a potent, tunable, broadly applicable and well-tolerable platform for targeted drug delivery and improved anticancer therapy.

A New Class of Tunable Acid-Sensitive Linkers for Native Drug Release Based on the Trityl Protecting Group.

Core-cross-linked polymeric micelles (CCPMs) are a promising nanoparticle platform due to favorable properties such as their long circulation and tumor disposition exploiting the enhanced permeability and retention (EPR) effect. Sustained release of covalently linked drugs from the hydrophobic core of the CCPM can be achieved by a biodegradable linker that connects the drug and the core. This study investigates the suitability of trityl-based linkers for the design of acid-triggered native active pharmaceutical ingredient (API) release from CCPMs. Trityl linker derivatives with different substituent patterns were synthesized and conjugated to model API compounds such as DMXAA-amine, doxorubicin, and gemcitabine, and their release kinetics were studied. Hereafter, API release from CCPMs based on mPEG-b-pHPMAmLac block copolymers was investigated. Variation of the trityl substitution pattern showed tunability of the API release rate from the trityl-based linker with t1/2 varying from <1.0 to 5.0 h at pH 5.0 and t1/2 from 6.5 to >24 h at pH 7.4, all at 37 °C. A clear difference in release kinetics was found between gemcitabine and doxorubicin, with gemcitabine showing no detectable release for 72 h at pH 5.0 and doxorubicin showing a t1/2 of less than 1 h. Based on these findings, we show that the reaction mechanism of trityl deprotection plays an important role in the API release kinetics. The first step in this mechanism, which is protonation of the trityl-bound amine, is pKa-dependent, which explains the difference in release rate. In conclusion, acid-sensitive and tunable trityl linkers are highly promising for the design of linker-API conjugates and for their use in CCPMs.

Utility of Intravenous Curcumin Nanodelivery Systems for Improving In Vivo Pharmacokinetics and Anticancer Pharmacodynamics.

Curcumin nanoformulations for intravenous injection have been developed to offset poor absorption, biotransformation, degradation, and excessive clearance associated with parenteral delivery. This review investigates (1) whether intravenous nanoformulations improve curcumin pharmacokinetics (PK) and (2) whether improved PK yields greater therapeutic efficacy. Standard PK parameters (measured maximum concentration [Cmax], area under the curve [AUC], distribution volume [Vd], and clearance [CL]) of intravenously administered free curcumin in mice and rats were sourced from literature and compared to curcumin formulated in nanoparticles, micelles, and liposomes. The studies that also featured analysis of pharmacodynamics (PD) in murine cancer models were used to determine whether improved PK of nanoencapsulated curcumin resulted in improved PD. The distribution and clearance of free and nanoformulated curcumin were very fast, typically accounting for >80% curcumin elimination from plasma within 60 min. Case-matched analysis demonstrated that curcumin nanoencapsulation generally improved curcumin PK in terms of measured Cmax (n = 27) and AUC (n = 33), and to a lesser extent Vd and CL. However, when the data were unpaired and clustered for comparative analysis, only 5 out of the 12 analyzed nanoformulations maintained a higher relative curcumin concentration in plasma over time compared to free curcumin. Quantitative analysis of the mean plasma concentration of free curcumin versus nanoformulated curcumin did not reveal an overall marked improvement in curcumin PK. No correlation was found between PK and PD, suggesting that augmentation of the systemic presence of curcumin does not necessarily lead to greater therapeutic efficacy.

Modulating albumin-mediated transport of peptide-drug conjugates for antigen-specific Treg induction.

The therapeutic potential of antigen-specific regulatory T cells (Treg) has been extensively explored, leading to the development of several tolerogenic vaccines. Dexamethasone-antigen conjugates represent a prominent class of tolerogenic vaccines that enable coordinated delivery of antigen and dexamethasone to target immune cells. The importance of nonspecific albumin association towards the biodistribution of antigen-adjuvant conjugates has gained increasing attention, by which hydrophobic and electrostatic interactions govern the association capacity. Using an ensemble of computational and experimental techniques, we evaluate the impact of charged residues adjacent to the drug conjugation site in dexamethasone-antigen conjugates (Dex-K/E4-OVA323, K: lysine, E: glutamate) towards their albumin association capacity and induction of antigen-specific Treg. We find that Dex-K4-OVA323 possesses a higher albumin association capacity than Dex-E4-OVA323, leading to enhanced liver distribution and antigen-presenting cell uptake. Furthermore, using an OVA323-specific adoptive-transfer mouse model, we show that Dex-K4-OVA323 selectively upregulated OVA323-specific Treg cells, whereas Dex-E4-OVA323 exerted no significant effect on Treg cells. Our findings serve as a guide to optimize the functionality of dexamethasone-antigen conjugate amid switching vaccine epitope sequences. Moreover, our study demonstrates that moderating the residues adjacent to the conjugation sites can serve as an engineering approach for future peptide-drug conjugate development.

Utilizing in vitro drug release assays to predict in vivo drug retention in micelles.

In the present work, we aim at developing an in vitro release assay to predict circulation times of hydrophobic drugs loaded into polymeric micelles (PM), upon intravenous (i.v.) administration. PM based on poly (ethylene glycol)-b-poly (N-2-benzoyloxypropyl methacrylamide) (mPEG-b-p(HPMA-Bz)) block copolymer were loaded with a panel of hydrophobic anti-cancer drugs and characterized for size, loading efficiency and release profile in different release media. Circulation times in mice of two selected drugs loaded in PM were evaluated and compared to the in vitro release profile. Release of drugs from PM was evaluated over 7 days in PBS containing Triton X-100 and in PBS containing albumin at physiological concentration (40 g/L). The results were utilized to identify crucial molecular features of the studied hydrophobic drugs leading to better micellar retention. For the best and the worst retained drugs in the in vitro assays (ABT-737 and BCI, respectively), the circulation of free and entrapped drugs into PM was examined after i.v. administration in mice. We found in vivo drug retention at 24 h post-injection similar to the retention found in the in vitro assays. This demonstrates that in vitro release assay in buffers supplemented with albumin, and to a lesser degree Triton X-100, can be employed to predict the in vivo circulation kinetics of drugs loaded in PM. Utilizing media containing acceptor molecules for hydrophobic compounds, provide a first screen to understand the stability of drug-loaded PM in the circulation and, therefore, can contribute to the reduction of animals used for circulation kinetics studies.

Tuning Surface Charges of Peptide Nanofibers for Induction of Antigen-Specific Immune Tolerance: An Introductory Study.

Induction of antigen-specific immune tolerance has emerged as the next frontier in treating autoimmune disorders, including atherosclerosis and graft-vs-host reactions during transplantation. Nanostructures are under investigation as a platform for the coordinated delivery of critical components, i.e., the antigen epitope combined with tolerogenic agents, to the target immune cells and subsequently induce tolerance. In the present study, the utility of supramolecular peptide nanofibers to induce antigen-specific immune tolerance was explored. To study the influence of surface charges of the nanofibers towards the extent of the induced immune response, the flanking charge residues at both ends of the amphipathic fibrillization peptide sequences were varied. Dexamethasone, an immunosuppressive glucocorticoid drug, and the ovalbumin-derived OVA323-339 peptide that binds to I-A(d) MHC Class II were covalently linked at either end of the peptide sequences. It was shown that the functional extensions did not alter the structural integrity of the supramolecular nanofibers. Furthermore, the surface charges of the nanofibers were modulated by the inclusion of charged residues. Dendritic cell culture assays suggested that nanofiber of less negative ζ-potential can augment the antigen-specific tolerogenic response. Our findings illustrate a molecular approach to calibrate the tolerogenic response induced by peptide nanofibers, which pave the way for better design of future tolerogenic immunotherapies.

Tuning the size of all-HPMA polymeric micelles fabricated by solvent extraction.

The size of polymeric micelles crucially affects their tumor accumulation, penetration and antitumor efficacy. In the present study, micelles were formed based on amphiphilic poly(N-2-hydroxypropyl methacrylamide)-block-poly(N-2-benzoyloxypropyl methacrylamide) (p(HPMAm)-b-p(HPMAm-Bz)) via the solvent extraction method, and factors impacting micelle size were systematically studied, including the molecular weight of the polymers, homopolymer content, and processing methods (i.e., batch process versus continuous microfluidics). The formation of core-shell structured micelles was demonstrated by light scattering, sedimentation velocity and electron microscopy analysis. Micellar size and aggregation number increased with decreasing the molecular weight ratio of the hydrophilic/hydrophobic block. The presence of hydrophobic p(HPMAm-Bz) homopolymer and high copolymer concentration increased micelle size, while the presence of hydrophilic p(HPMAm) homopolymer did not affect micellar size. Regarding processing conditions, it was found that the use of tetrahydrofuran and acetone as solvents for the polymers resulted in larger micelles, likely due to their relatively high water-solvent interaction parameters as compared to other solvents tested, i.e., dimethylformamide, dimethylacetamide, and dimethyl sulfoxide. Among the latter, only dimethylformamide led to micelles with a narrow polydispersity. Addition of dimethylformamide to an aqueous solvent and faster mixing of two solvents using microfluidics favored the formation of smaller micelles. In conclusion, our results show that the size of all-HPMA polymeric micelles can be easily tailored from 40 to 120 nm by varying the formulation properties and processing parameters.

Oral pretreatment with ß-lactoglobulin derived peptide and CpG co-encapsulated in PLGA nanoparticles prior to sensitizations attenuates cow's milk allergy development in mice.

Cow's milk allergy is a common food allergy among infants. Improved hygiene conditions and loss of microbial diversity are associated with increased risk of allergy development. The intestinal immune system is essential for oral tolerance induction. In this respect, bacterial CpG DNA is known to drive Th1 and regulatory T-cell (Treg) development via Toll-Like-Receptor 9 (TLR-9) signaling, skewing away from the allergic Th2 phenotype. We aimed to induce allergen specific tolerance via oral delivery of poly (lactic-co-glycolic acid) nanoparticles (NP) co-encapsulated with a selected ß-lactoglobulin derived peptide (BLG-Pep) and TLR-9 ligand CpG oligodeoxynucleotide (CpG). In vivo, 3-4-week-old female C3H/HeOuJ mice housed in individually ventilated cages received 6-consecutive-daily gavages of either PBS, whey, BLG-Pep/NP, CpG/NP, a mixture of BLG-Pep/NP plus CpG/NP or co-encapsulated BLG-Pep+CpG/NP, before 5-weekly oral sensitizations with whey plus cholera toxin (CT) or only CT (sham) and were challenged with whey 5 days after the last sensitization. The co-encapsulated BLG-Pep+CpG/NP pretreatment, but not BLG-Pep/NP, CpG/NP or the mixture of BLG-Pep/NP plus CpG/NP, prevented the whey-induced allergic skin reactivity and prevented rise in serum BLG-specific IgE compared to whey-sensitized mice. Importantly, co-encapsulated BLG-Pep+CpG/NP pretreatment reduced dendritic cell (DC) activation and lowered the frequencies of PD-L1+ DC in the mesenteric lymph nodes compared to whey-sensitized mice. By contrast, co-encapsulated BLG-Pep+CpG/NP pretreatment increased the frequency of splenic PD-L1+ DC compared to the BLG-Pep/NP plus CpG/NP recipients, in association with lower Th2 development and increased Treg/Th2 and Th1/Th2 ratios in the spleen. Oral administration of PLGA NP co-encapsulated with BLG-Pep and CpG prevented rise in serum BLG-specific IgE and symptom development while lowering splenic Th2 cell frequency in these mice which were kept under strict hygienic conditions.

Preparation of mRNA Polyplexes with Post-conjugated Endosome-Disruptive Peptides.

Successful delivery of mRNA into the cytosol of professional antigen-presenting cells (APCs) poses one of the biggest challenges in developing effective mRNA vaccines to treat various cancers and viral infectious diseases. However, most polymeric mRNA delivery systems fail to transfect APCs. We have discovered that decoration of pH-sensitive endosome-disruptive GALA peptides on the surface of mRNA polyplexes leads to efficient targeting and transfection of APCs. GALA peptides not only enhance specific uptake in APCs through binding to sialic acid moieties, they also facilitate the endosomal escape of mRNA especially in dendritic cells (DCs). Here, we describe in detail the production of stabilized mRNA polyplexes post-conjugated with GALA peptides via copper-free click chemistry. Methods described here include the synthesis and purification of GALA peptides and its conjugation to mRNA polyplexes.

Molecular targets for anticancer therapies in companion animals and humans: what can we learn from each other?

Despite clinical successes in the treatment of some early stage cancers, it is undeniable that novel and innovative approaches are needed to aid in the fight against cancer. Targeted therapies offer the desirable feature of tumor specificity while sparing healthy tissues, thereby minimizing side effects. However, the success rate of translation of these therapies from the preclinical setting to the clinic is dramatically low, highlighting an important point of necessary improvement in the drug development process in the oncology field. The practice of a comparative oncology approach can address some of the current issues, by introducing companion animals with spontaneous tumors in the linear drug development programs. In this way, animals from the veterinary clinic get access to novel/innovative therapies, otherwise inaccessible, while generating robust data to aid therapy refinement and increase translational success. In this review, we present an overview of targetable membrane proteins expressed in the most well-characterized canine and feline solid cancers, greatly resembling the counterpart human malignancies. We identified particular areas in which a closer collaboration between the human and veterinary clinic would benefit both human and veterinary patients. Considerations and challenges to implement comparative oncology in the development of anticancer targeted therapies are also discussed.