Cleavage Of Disulfide Linkages Research Articles

Use of a novel proteomimetic nanoplatform for simultaneous delivery of antigens with adjuvants to impact tumor growth in multiple tumor models.

40 Background: Patient-specific cancer vaccines derived from tumor antigen have been explored as a promising therapeutic strategy, however, challenges delivering vaccine components in a coordinated fashion to elicit antitumor responses remain. To overcome these, we utilize a novel nanoplatform called the Protein-Like Polymer (PLP), which allows for sustained and targeted delivery of tumor antigens in conjunction with adjuvants. Methods: PLPs containing peptide antigens were synthesized via ROMP and characterized. A library of compounds with different sidechain linkage chemistries, degrees of polymerization (DP), and inclusion/exclusion of Oligo-ethylene glycol (OEG) were made to determine design rules for immune activation. Cell uptake and functional assays using payload-specific T Cells were conducted. Immunization in three independent tumor models was done to show generalizability. Ability of PLPs to co-deliver adjuvants was tested by electrostatically coupling small molecule STING agonist, 2’3’ cGAMP. Results: Conjugating antigens to the polymer via a cleavable disulfide linkage, which reduces intracellularly in APCs, resulted in increased endosomal localization, higher levels of induced T cell proliferation, cytokine production, and expression of activation markers in CTLs and APCs. Incorporating a diluent amount of OEG side chains reduced enzymatic degradation while increasing immunogenicity and uptake. Additionally, increasing the DP, and therefore the density of antigen side chains, further improved vaccine efficacy and resistance to proteolysis. Antigen-PLP conjugates enhanced dendritic cell activation and T-cell response only when paired with cells from their cognate system, with no activity in immune cells not expressing receptors for the payload demonstrating antigen-specificity. Mice bearing established B16F10, MC38, or TC-1 tumors treated with PLPs containing gp100, adpgk, or E7 respectively all showed increased survival times, reduced tumor burden with corresponding changes in immune cell profiles, and immunological memory upon rechallenge. Impressively, mice treated with STING-PLP complexes had significantly smaller tumors vs control at day 14 (0.038g vs 0.76g; p < 0.0001) and allowed for subcutaneous administration of 2’3’ cGAMP, which traditionally requires intratumoral injection. Studies on the effects of vaccinating with pools of neoantigens multiplexed onto one PLP are ongoing. Conclusions: This work validates the ability of PLPs to overcome major limitations in cancer vaccine development. The modularity of the platform allows for complex nano-architectures including systems capable of delivering challenging compounds, ie small molecule STING agonists, subcutaneously through electrostatic coupling, highlighting its potential to revolutionize cancer vaccinology.

Fluoro-Crown Ether Phosphate as Efficient Cell-Permeable Drug Carrier by Disrupting Hydration Layer.

Fast and direct permeation of drug molecules is crucial for effective biotherapeutics. Inspired by a recent finding that fluorous compounds disrupt the hydrogen-bonded network of water, we developed fluoro-crown ether phosphate CyclicFP-X. This compound acts as a fast cell-permeating agent, enabling direct delivery of various bioactive cargos (X) into cancer cells without endocytic entrapment. In contrast, its nonfluorinated cyclic analog (CyclicP-X) failed to achieve cellular internalization. Although the acyclic fluorous analog AcyclicFP-X was internalized, this process occurred slowly owing to the involvement of an endocytic trapping pathway. Designed with a high fluorine density, CyclicFP-X exhibits compactness, polarity, and high-water solubility, facilitating lipid vesicle fusion by disrupting their hydration layers. Raman spectroscopy confirmed the generation of dangling -OH bonds upon addition of CyclicFP-OH to water. Furthermore, conjugating CyclicFP-X with fluorouracil (FU, an anticancer drug) via a reductively cleavable disulfide linker (CyclicFP-SS-FU) demonstrated the general utility of fluoro-crown ether phosphate as a potent carrier for biotherapeutics.

Abstract 481: Proteomimetic polymers limit tumor growth in multiple models via potent antigen-specific T cell activation

Abstract Background: Patient-specific cancer vaccines derived from tumor antigen have been explored as a promising therapeutic strategy, however, challenges delivering vaccine components in a coordinated fashion to elicit antitumor responses remain. To overcome these, we utilize a novel nanoplatform called the Protein-Like Polymer (PLP), which allows for sustained and targeted delivery of tumor antigens in conjunction with adjuvants. Methods: PLPs containing peptide antigens were synthesized via ring-opening metathesis polymerization (ROMP) and characterized. A library of compounds with different sidechain linkage chemistries, degrees of polymerization (DP), and inclusion/exclusion of Oligo-ethylene glycol (OEG) were made to determine design rules for immune activation. Cell uptake and functional assays using payload-specific T Cells were conducted. Immunization in three independent tumor models was done to show generalizability. Ability of PLPs to co-deliver adjuvants was tested by electrostatically coupling small molecule STING agonist, 2’3’ cGAMP. Results: Conjugating peptide antigens to the polymer via a cleavable disulfide linkage, which reduces intracellularly in APCs, resulted in increased endosomal localization, higher levels of induced T cell proliferation, cytokine production, and expression of activation markers in CTLs and APCs. Incorporating a diluent amount of OEG side chains reduced enzymatic degradation while increasing immunogenicity and uptake. Additionally, increasing the DP, and therefore the density of antigen side chains, further improved vaccine efficacy and resistance to proteolysis. Antigen-PLP conjugates enhanced dendritic cell activation and T-cell response only when paired with cells from their cognate system, with no activity in immune cells not expressing receptors for the payload demonstrating antigen-specificity. Mice bearing established B16F10, MC38, or TC-1 tumors treated with PLPs containing gp100, adpgk, or E7 respectively all showed increased survival times, reduced tumor burden with corresponding changes in immune cell profiles, and immunological memory upon rechallenge. Impressively, mice treated with STING-PLP complexes had significantly smaller tumors vs control at day 14 (0.038g vs 0.76g; p <0.0001) and allowed for subcutaneous administration of 2’3’ cGAMP, which traditionally requires intratumoral injection. Studies on the effects of vaccinating with pools of neoantigens multiplexed onto one PLP are ongoing. Conclusion: This work validates the ability of PLPs to overcome major limitations in cancer vaccine development. The modularity of the platform allows for complex nano-architectures including systems capable of delivering challenging compounds, ie small molecule STING agonists, subcutaneously through electrostatic coupling, highlighting its potential to revolutionize cancer vaccinology. Citation Format: Max Wang, Miran Choi, Bin Zhang, Nathan Gianneschi. Proteomimetic polymers limit tumor growth in multiple models via potent antigen-specific T cell activation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 481.

Use of proteomimetic polymers for delivery of tumor antigens and adjuvants through formation of stable electrostatic complexes with small molecule STING agonists.

2583 Background: Utilizing tumor antigens for the development of patient-specific cancer vaccines has been a promising therapeutic strategy. However, significant challenges remain in delivering subunit vaccine components to elicit antitumor immune responses. To overcome these, we developed rationally designed cancer vaccines using a new nanoplatform called the Protein-Like Polymer (PLP) with unique characteristics that allow for sustained delivery of tumor antigens in conjunction with immunomodulatory compounds. Methods: PLPs containing antigens were synthesized via ring-opening metathesis polymerization (ROMP) and characterized using HPLC, LC-MS, NMR, and GPC. A library of compounds were generated with different sidechain linkage chemistries (amide, ester, or disulfide linkage), DPs, and inclusion of PEG side chains. In vitro assays using gp100-specific T Cells were conducted with fluorescently labeled and non-labeled polymers. In vivo experiments were done using B16 murine melanoma transplanted tumor models. Ability of PLPs to co-deliver immunomodulatory compounds was tested using a small molecule STING agonist 2’3’ cGAMP. Results: Conjugating antigens to the polymer backbone using a cleavable disulfide linkage, designed to be reduced intracellularly, resulted in the best subcellular localization and immune activation (higher levels of immune cell proliferation, cytokine production, and expression of activation markers). Incorporating a diluent amount of PEG side chains increased resistance to enzymatic degradation while improving bioactivity and cell uptake. In vivo studies using PLPs conjugated with melanoma-associated antigen gp100 showed the reductions in tumor size with increased immune cell proliferation and activation in a B16 melanoma model. Additionally, increasing the degree of polymerization, and therefore the density of antigen side chains per unit polymer, improved vaccine efficacy and resistance to proteolysis. Mice treated with STING agonists electrostatically coupled to PLPs showed significantly smaller tumors vs no-treatment at day 14 (0.038g vs 0.76g; p <0.0001) and allowed for subcutaneous administration of the small molecule, which normally must be injected intratumorally. Conclusions: Our work demonstrates the potential of PLPs to overcome inherent difficulties associated with developing cancer vaccines. The modularity of the platform allows for complex nano-architectures including systems capable of delivering challenging compounds, ie small molecule STING agonists, subcutaneously through electrostatic coupling. This technology has the potential to revolutionize cancer vaccinology.

Design and Optimization of a Novel Proteomimetic Polymer Nanoplatform for the Co-delivery of Antigens with Adjuvants

Abstract We describe here a novel nanoplatform called the Protein-Like Polymer (PLP) with unique characteristics allowing for sustained delivery of tumor antigens in conjunction with STING agonists. Methods: A library of PLP formulations were synthesized via ROMP and characterized. Cell uptake/functional assays were conducted with payload-specific T Cells. Ability of PLPs to co-deliver adjuvants was tested by electrostatically coupling 2′3′ cGAMP, forming stable nanostructures. Results: Conjugating antigens to the polymer backbone using a cleavable disulfide linkage (reduces in APCs) resulted in increased endosomal localization and T cell proliferation/activation. Incorporating OEG side chains reduced enzymatic degradation while increasing immunogenicity and APC uptake. Increasing the density of side chains further improved vaccine efficacy and resistance to proteolysis. Antigen-PLP conjugates is effective only when paired with cells from their cognate system, demonstrating payload-specificity. Mice bearing established B16F10 tumors treated with gp100-PLPs resulted in increased survival time and reduced tumor burden with corresponding changes in immune cell profiles. Notably, STING-PLP complexes showed significantly smaller tumors at day 14 and allowed for subcutaneous administration of 2′3′ cGAMP. Studies looking at multiplexing pools of neoantigens onto one PLP and generation of immunological memory are ongoing. Conclusion: The modularity of the PLP platform allows for complex nano-architectures including systems capable of delivering challenging compounds, ie small molecule STING agonists, subcutaneously through electrostatic coupling, highlighting its potential to revolutionize cancer vaccinology. Supported by grants from the NIH (5F30CA257519-03)

A Cleavable Self-Inclusion Conjugate with Enhanced Biocompatibility and Antitumor Bioactivity

A Cleavable Self-Inclusion Conjugate with Enhanced Biocompatibility and Antitumor Bioactivity

A Fluorescence Study on Guest Release of Reduction-responsive Host-guest Conjugates Having a Disulfide Bond

Reduction-responsive and anionic cyclophane (1) having a fluorescent pyrene moiety with a cleavable disulfide linkage was synthesized as a host-guest conjugate covalently bound with both host (cyclophane) and guest (pyrene) components. Reduction of the disulfide bond of 1 by glutathione gave its reduced forms (thiolates) of cyclophane and released pyrene derivatives that led to a significant fluorescence response in an aqueous carbonate buffer. The fluorescence intensity originating from monomer emission of 1 considerably decreased, accompanying increase of excimer emission due to self-aggregation of the resulting amphiphilic thiolate of pyrene derivative.

A novel polymeric peptide delivery platform and association with targeted co-delivery of antigens and STING agonists with antitumor immune response.

2564 Background: Use of tumor antigens for the development of patient-specific cancer vaccines has been a promising therapeutic strategy. However, challenges remain in delivering subunit vaccine components in a coordinated fashion to elicit antitumor immune responses. To overcome these, we developed rationally designed vaccines using a novel nanoplatform called the Protein-Like Polymer (PLP), in reference to its globular structure reminiscent of native proteins, with unique characteristics that allow for sustained/targeted delivery of tumor antigens in conjunction with STING agonists. Methods: PLPs containing a model melanoma antigen were synthesized via ring-opening metathesis polymerization (ROMP) and characterized. A library of compounds were generated with different sidechain linkage chemistries (amide, ester, or disulfide), degrees of polymerization, and inclusion/exclusion of Oligo(ethylene glycol) (OEG) side chains. In vitro uptake and functional assays using gp100-specific T Cells were conducted with fluorescently-labeled and non-labeled polymers respectively. In vivo experiments were done using a B16F10 murine melanoma tumor model over-expressing gp100. Ability of PLPs to co-deliver immunomodulatory compounds was tested by electrostatically coupling a small molecule STING agonist 2’3’ cGAMP which formed stable nanostructures. The optimized construct was also tested in an OVA system to prove generalizability. Results: Conjugating peptide antigens using a cleavable disulfide linkage, which reduces intracellularly in antigen presenting cells (APCs), resulted in increased endosomal localization and efficacy. Incorporating a diluent amount of OEG side chains increased resistance to enzymatic degradation while improving bioactivity and uptake by APCs. In vivo studies using PLPs conjugated with gp100 resulted in significant increases in survival time and reduced tumor burden in B16 melanoma. Increasing the DP, and therefore the density of antigen side chains, improved vaccine efficacy and resistance to proteolysis. Mice treated with STING-PLP complexes showed significantly smaller tumors vs control at day 14 (0.038g vs 0.76g; p < 0.0001) and allowed for subcutaneous administration of 2’3’ cGAMP, which otherwise diffuses rapidly away from the injection site. OVA-PLPs behaved similarly in their cognate system but showed no activity when tested on gp100-specific cells and vice versa demonstrating antigen-specificity. Conclusions: This work validates the potential of PLPs to overcome major limitations in cancer vaccine development. The modularity of the platform allows for complex nano-architectures including systems capable of delivering challenging compounds, ie small molecule STING agonists, subcutaneously through electrostatic coupling. This technology has the potential to revolutionize cancer vaccinology.

A Redox-Activatable and Targeted Photosensitizing Agent to Deliver Doxorubicin for Combining Chemotherapy and Photodynamic Therapy

Currently, tumors have become a serious disease threatening human health and life in modern society. Photo-chemo combination therapy is considered to be an important method to improving the efficiency of tumor treatment, especially in the treatment of multi-drug-resistant tumors. However, the application of photo-chemo combination therapy has been limited by the poor water solubility of photosensitizers, low tumor targeting, and high side effects of chemotherapy drugs. In order to solve these problems, a smart nano drug delivery platform FA-PEG-ss-PLL(-g-Ce6) designed and synthesized by us. The smart nano drug carrier uses folic acid (FA) as the targeting group, polyethylene glycol (PEG) as the hydrophilic end, Ce6-grafted polylysine (PLL(-g-Ce6)) as the hydrophobic end, and Chlorin e6 (Ce6) as the photosensitizer of photodynamic therapy, and it connects PEG to PLL by a redox-responsive cleavable disulfide linker (-ss-). Finally, the combination of tumor chemotherapy and photodynamic therapy (PDT) is realized by loading with anticancer drug doxorubicin (DOX) to the intelligent carrier. In vitro experiments showed that the drug loading content (DLC%) of DOX@FA-PEG-ss-PLL(-g-Ce6) nanoparticles (DOX@FPLC NPs) was as high as 14.83%, and the nanoparticles had good serum stability, reduction sensitivity and hemocompatibility. From the cytotoxicity assays in vitro, we found that under 664 nm laser irradiation DOX@FPLC NPs showed stronger toxicity to MCF-7 cells than did DOX, Ce6 + laser, and DOX + Ce6 + laser. Moreover, the antitumor efficiency in vivo and histopathological analysis showed that DOX@FPLC NPs under 664 nm laser irradiation exhibited higher antitumor activity and lower systemic toxicity than single chemotherapy. These results suggested that the FA-PEG-ss-PLL(-g-Ce6) nano drug delivery platform has considerable potential for the combination of chemotherapy and PDT.

Synthesis of a Degradable Hydrogel Based on a Graft Copolymer with Unexpected Thermoresponsiveness

AbstractIncorporating multiple pyridyl disulfide (PDS) moieties into polymer chains allows the fabrication of a chemically cross‐linked hydrogel through the rapid thiol–disulfide exchange reaction. By aminolysis in the presence of 2,2′‐dithiodipyridine (DTP), the end groups of polymers synthesized via reversible addition−fragmentation chain transfer (RAFT) polymerization can readily be converted into PDS‐groups. In this contribution, a RAFT‐synthesized graft copolymer with thermoresponsive poly[di(ethylene glycol) methyl ether methacrylate] forming the backbone and hydrophilic poly(N,N‐dimethylacrylamide) as the side chains is presented. The copolymer chains exhibit surprisingly a two‐step lower critical solution temperature transition in aqueous solutions. After modification of the end groups of the backbone and side chains, the PDS‐terminated chains can react with a dithiol cross‐linker to form a thermoresponsive hydrogel. In a reducing environment, the cleavable disulfide linkages lead to on‐demand dissolution of the hydrogel. The resulting thiol‐terminated chains undergo a reversible sol–gel transition in response to redox variations, expanding the potential application areas of such a polymer system.

Synthesis and Self‐Assembly of Stimuli‐Responsive Amphiphilic Dendrimers

AbstractThe synthesis of stimuli‐responsive amphiphilic dendrimers consisting of two or three hydrophobic octadecyl groups linked to either a second or third generation hydrophilic polylysine dendron through disulfide linkage is reported. The polylysine dendrimers functionalized with four or eight tert‐butyloxycarbonyl (Boc)‐protected amino groups are characterized by 1H NMR spectroscopy and matrix‐assisted laser desorption ionization time‐of‐flight (MALDI‐TOF) mass spectrometry. With the positively charged versions generated by removing the Boc groups, their ability to form spherical micelles that can be readily disassembled in the presence of glutathione (GSH) is demonstrated. The self‐assembly behavior is monitored by scanning electron microscopy (SEM) and dynamic light scattering (DLS). The amphiphilic dendritic systems containing cleavable disulfide linkage serve as key building blocks for generating stimuli‐responsive supramolecular architectures.

Synthesis and characterization of lysozyme-conjugated Ag.ZnO@HA nanocomposite: A redox and pH-responsive antimicrobial agent with photocatalytic activity

Hydroxyapatite (HA) is extensively used for implantable device coating; however, it lacks antibacterial property, leading to potential bacterial infection during orthopedic implantation surgery. Herein, to enhance the antibacterial activity of HA, a redox- and pH-responsive HA nanocomposite with photocatalytic activity was designed. A photosensitive heterostructure, zinc oxide/hydroxyapatite (ZnO.HA), was coated with Ag nanoparticles (AgNPs) with assisted gallic acid using the UV-irradiation method. An antibacterial enzyme, lysozyme, was then conjugated on the surface of the nanocomposite by a cleavable disulfide linker, resulting in a redox-sensitive nanoplatform. In comparison with bare HA, the designed nanocomposites as Lyso.CAGZ@HA displayed much higher antibacterial activity (> 5-fold) toward Escherichia coli (E. coli) owing to the synergistic antibacterial effects of ZnONPs, AgNPs, gallic acid, and lysozyme on the surface of the nanocomposite. However, antibacterial and antifouling effects are much more enhanced in Lyso.CAGZ@HA-treated bacteria as they were subjected to UVA irradiation. Moreover, the cellular uptake of nanocomposite and intracellular glutathione depletion enhanced in the presence of UVA light, resulting in reactive oxygen specious generation enhancement. Further, in vitro cytotoxicity experiments on mammalian cells (human foreskin fibroblast) revealed that nanocomposite has no cytotoxic effects. Hence, this study demonstrated that Lyso.CAGZ@HA could be considered as a potential therapeutic approach against bacterial infectious diseases.

A Dendron-Based Fluorescence Turn-On Probe for Tumor Detection.

Specifically amplifying the emission signals of optical probes in tumors is an effective way to improve the tumor-imaging sensitivity and contrast. In this paper, the first case of dendron-based fluorescence turn-on probes mediated by a Förster resonance energy transfer (FRET) mechanism is reported. Dendrons up to the fourth generation with a hydrophilic oligo(ethylene glycol) scaffold are synthesized by a solid-phase synthesis strategy, and show precise and defect-free chemical structures. To construct the fluorescence turn-on probe, one Cy5.5 molecule is conjugated to the focal of a G3 dendron through a robust linkage and eight Black Hole Quencher 3 (BHQ-3) molecules are conjugated to its periphery through a PEG chain bearing a reductively cleavable disulfide linkage. By in vitro and in vivo experiments, it is demonstrated that the fluorescence of the dendron-based probe can be activated effectively and rapidly in the reductive environments of tumor cells and tissues, and the probe thus exhibits amplified tumor signals and weak normal tissue signals. Compared with the reported nanoscale turn-on probes, the dendron-based probe has several significant advantages, such as well-defined chemical structure, precisely controllable fluorophore/quencher conjugation sites and ratio, desirable chemical stability, and reproducible pharmacokinetic and pharmacological profiles, and is very promising in tumor detection.

Reversible Functionalization of Clickable Polyacrylamide Gels with Protein and Graft Copolymers.

Modular strategies to fabricate gels with tailorable chemical functionalities are relevant to applications spanning from biomedicine to analytical chemistry. Here, the properties of clickable poly(acrylamide-co-propargyl acrylate) (pAPA) hydrogels are modified via sequential in-gel copper-catalyzed azide-alkyne cycloaddition (CuAAC) reactions. Under optimized conditions, each in-gel CuAAC reaction proceeds with rate constants of ~0.003 s-1, ensuring uniform modifications for gels < 200 μm thick. Using the modular functionalization approach and a cleavable disulfide linker, pAPA gels were modified with benzophenone and acrylate groups. Benzophenone groups allow gel functionalization with unmodified proteins using photoactivation. Acrylate groups enabled copolymer grafting onto the gels. To release the functionalized unit, pAPA gels were treated with disulfide reducing agents, which triggered ~50 % release of immobilized protein and grafted copolymers. The molecular mass of grafted copolymers (~6.2 kDa) was estimated by monitoring the release process, expanding the tools available to characterize copolymers grafted onto hydrogels. Investigation of the efficiency of in-gel CuAAC reactions revealed limitations of the sequential modification approach, as well as guidelines to convert a pAPA gel with a single functional group into a gel with three distinct functionalities. Taken together, we see this modular framework to engineer multifunctional hydrogels as benefiting applications of hydrogels in drug delivery, tissue engineering, and separation science.

Stimuli-Responsive Supramolecular Coaggregation and Disaggregation of Host-Guest Conjugates Having a Disulfide Linkage.

Water-soluble cationic and anionic cyclophanes (1a and 2a, respectively) having a dabsyl group with a cleavable disulfide linkage were synthesized as a host-guest conjugate covalently bound with both host and guest components. Self-inclusion phenomena but not self-aggregation behaviors were observed for each cyclophane in aqueous media. Each cyclophane includes its own dabsyl moiety (guest component) in its macrocyclic cavity (host component) through hydrophobic interaction. When 1 equiv. of cationic 1a was added to an aqueous solution of anionic 2a, however, supramolecular coaggregates formed spontaneously through host-guest complexation. As regard the supramolecular coaggregates, the existence of larger particles was confirmed by DLS measurements and TEM observation. The hydrophobic interaction between the dabsyl moiety and macrocyclic cavity and electrostatic interactions between 1a and 2a play important roles in the supramolecular coaggregate formation. Each cyclophane having a cleavable disulfide linkage was easily transformed to the corresponding thiols by reducing reagents such as DTT, which was confirmed by MALDI-TOF MS. Disaggregation of the supramolecular coaggregates composed of 1a and 2a was successfully performed upon addition of DTT, with release of the thiol derivative of dabsyl. Such disaggregation of the coaggregates was also conducted by other external stimuli such as salts and competitive guests.

Nanoapplication of a Resistance Inducer to Reduce Phytophthora Disease in Pineapple (Ananas comosus L.).

Treatment of plants with a variety of abiotic and biotic inducers causes induced resistance to pathogen attack. In this study, the effect of four resistance inducers on plant diseases caused by Phytophthora cinnamomi was screened in vivo initially by using lupin, a susceptible model plant. Lupin pretreated with 0.5 mM salicylic acid (SA) showed effective resistance against P. cinnamomi with restricted lesions. Then, mesoporous silica nanoparticles (MSNs) with particle size around 20 nm and approximate pore size of 3.0 nm were synthesized and functionalized for loading and importing SA to pineapple plantlets. Decanethiol gatekeepers were introduced to the surface of MSNs via glutathione (GSH)–cleavable disulfide linkages to cover the pore entrance, which was confirmed through using Raman spectroscopy. Through free diffusion, the loading efficiency of SA in MSNs gated with gatekeepers was 11.7%, but was lower in MSNs without gatekeepers (8.0%). In addition, in vitro release profile of SA from gatekeeper-capped MSNs indicated that higher concentrations of GSH resulted in more cargo release. Moreover, the experiments in planta showed that the application of MSNs as a resistance inducer delivery system significantly improved pineapple resistance to P. cinnamomi in terms of inhibiting lesion development and improving root growth of infected plants, compared to the use of free SA and MSNs without gatekeepers. The analysis of SA, GSH, and defense-related genes, of PR1 and PR5, further confirmed that the slow and prolonged release of SA from MSNs inside the roots of pineapple plants was achieved through a redox-stimuli release mechanism. Therefore, the application of MSNs with redox-responsive gatekeepers has shown great potential as an efficient tool for delivering chemicals into plants in a controllable way.

Challenges to Achieving Strong but Fully Degradable Adhesive Joints between Wet Cellulose Surfaces.

The dramatic loss of strength upon exposure to water is one of the challenges preventing the widespread substitution of plastic packaging with paper and paperboard. Although treatment with conventional wet strength resins and other adhesive polymers can strengthen wet paper, it is at the expense of green credentials, including easy recycling or rapid composting. The goal of this work was to demonstrate the adhesive requirements for strong, wet cellulose-cellulose joints that can be recycled easily because the joint strength can be destroyed by the presence of a weak reducing agent. Cellulose membrane surfaces were first treated with a bound layer of carboxymethyl cellulose, modified to have covalently tethered hydrazide groups. Joints were fabricated by laminating two hydrazide-modified membranes with a polymeric adhesive bearing aldehyde functionality. Aldehydes spontaneously condense with hydrazide to give hydrazone bonds. When the adhesive was oxidized dextran, the wet laminates had an intermediate strength. Upon exposure to a reducing agent, the joint strength was reduced to nearly zero because every hydrazone moiety connecting two cellulose surfaces included a cleavable disulfide linkage. By contrast, glyoxalated cationic polyacrylamide gave very strong wet joints. However, the wet adhesion was dominated by polyelectrolyte complexation, and the presence of hydrazone linkages had little influence on the wet adhesion. We conclude that robust joint degradability will require cleavable linkages in the adhesive backbone while minimizing polyelectrolyte complexation.

Abstract 4328: Epigenetic modulation of SSTR2 expression provides the potential to broaden PEN-221 treatment population

Abstract The expression of somatostatin receptor subtype 2 (SSTR2) is increased in up to 80% of neuroendocrine tumors (NETs), 20-40% of small cell lung cancers (SCLC) and a number of other tumor types. As a cell surface receptor, SSTR2 is a logical target for a drug conjugate. However, marginal, or loss of, expression of SSTR2 in tumor tissues has potential to limit the benefit from such conjugates. Recent published preclinical studies have demonstrated that epigenetic modulators, such as HDAC inhibitors, can result in increased SSTR2 expression with treatment. Given that the expression of SSTR2 can be variable from patient to patient and in some cases low to absent, we hypothesized that the combination of epigenetic modulators with an SSTR2 target therapy may provide a benefit for patients who are not eligible for these targeted approaches on their own. PEN-221 is a miniature drug conjugate of an SSTR2 targeting peptide attached to the potent cytotoxic DM1 through a cleavable disulfide linker. PEN-221, currently in Phase 1/2a (NCT02936323), is designed as a potent and selective anticancer agent to treat patients whose tumors express SSTR2. The targeting peptide of PEN-221 selectively binds to SSTR2, triggers receptor internalization leading to the accumulation of the DM1 to provide antitumor activity by disrupting microtubule networks causing apoptosis and mitotic catastrophe. In preclinical studies, single agent PEN-221 treatment leads to complete and sustained tumor regression in xenograft models that over-express SSTR2. We hypothesize that increased expression of SSTR2 as a result of epigenetic modulation from HDAC or other epigenetic modulators, may allow for the broadening of PEN-221 treatable tumors. To evaluate these hypotheses, we have combined PEN-221 and epigenetic modulators in preclinical models of cancer. Efficacy studies were carried out in models expressing various degrees of SSTR2, and combinations resulted in greater efficacy than that of the single agent. In addition, a pharmacodynamic assessment of epigenetic modulator treatment on SSTR2 expression levels was performed. These data demonstrate that combination of PEN-221 and epigenetic modulators provide greater efficacy than single agent activity alone and support the evaluation of such combinations in the clinical setting. Citation Format: Samantha Perino, James Quinn, Jessica Freda, Brian White, Scott Bailey, Viren Bhatia, Beata Sweryda-Krawiec, Mark Bilodeau, Jeffrey D. Bloss, Kerry Whalen, Richard Wooster. Epigenetic modulation of SSTR2 expression provides the potential to broaden PEN-221 treatment population [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4328.

Dimethyl 3,3′-dithiobispropionimidate (DTBP) as a cleavable disulfide-based polymer to encapsulate nucleic acids in biological sample preparation

Cleavable disulfide bonds are extremely valuable functional groups for numerous biological applications, including applications as antitumor treatments, gene delivery, and diagnosis. Herein, we demonstrate the use of dimethyl 3,3′-dithiobispropionimidate (DTBP) as a disulfide cleavable bond based cross-linker in nano-composites that encapsulate and release nucleic acids in sample preparation systems for disease diagnoses. We conjugated DTBP with nano-composites, such as amine group-modified diatomaceous earth (DA), which is a solid substrate, and a zinc oxide nanomultigonal shuttle (ZnO NMS) that disrupts cell membranes without chaotropic detergents and improved the quantity and purity of nucleic acids from various biological samples. After encapsulating nucleic acids, the cleavable disulfide bond of DTBP was used to release nucleic acids by breaking disulfide bonds using dithiothreitol (DTT), resulting in highly enhanced efficiency of downstream analyses without PCR inhibitors such as high salt concentrations and SDS. Therefore, nano-composites with cleavable disulfide linkages have potential for various biological applications, including disease diagnostics and therapies.

Proactively Reducing Anti-Drug Antibodies via Immunomodulatory Bioconjugation.

Although PEGylation reduces the immunogenicity of protein drugs to some extent, its limitations for highly immunogenic biotherapeutics have been demonstrated. Herein, a proactive strategy to alleviate the development of anti-drug antibodies (ADAs) against protein drugs by immunomodulatory bioconjugation is reported. Rapamycin was conjugated to a PEGylated protein therapeutic via a cleavable disulfide linker. The conjugated rapamycin can be released from the bioconjugate and prevent immune responses once the bioconjugate is uptaken by antigen-presenting cells. The immunomodulatory bioconjugate significantly reduced the titers of ADAs compared with a PEGylated protein. The inhibition of immune responses was specific to the conjugated antigen, avoiding systemic immune suppression and the risk of increased susceptibility to infections. The reported approach breaks the limitations of PEGylation by the proactive prevention of ADAs.