Enzymatically-crosslinked gelatin hydrogels containing paenipeptin and clarithromycin against carbapenem-resistant pathogen in murine skin wound infection

Background The recent rise and spread of carbapenem-resistant pathogens pose an urgent threat to public health and has fueled the search for new therapies. Localized delivery of topical antibiotics is an alternative for the treatment of infected wounds caused by drug-resistant pathogens. In this study, we aimed to develop antimicrobial-loaded hydrogels for topical treatment of wound infections in a murine skin wound infection. Results Paenipeptin analogue 1, a linear lipopeptide, potentiated clarithromycin against multidrug-resistant Acinetobacter baumannii, Enterobacter cloacae, Escherichia coli, and Klebsiella pneumoniae. Enzymatically-crosslinked gelatin hydrogels were developed to encapsulate paenipeptin analogue 1 and clarithromycin. The encapsulated antimicrobials were gradually released from hydrogels during incubation, reaching 75.43 and 53.66% for paenipeptin and clarithromycin, respectively, at 24 h. The antimicrobial-loaded hydrogels containing paenipeptin and clarithromycin synergistically resulted in 5-log reduction in carbapenem-resistant A. baumannii within 6 h in vitro. Moreover, the antimicrobial-loaded hydrogels reduced 3.6- and 2.5-log of carbapenem-resistant A. baumannii when treated at 4 or 20 h post infection, respectively, in a murine skin wound infection. Conclusions Enzymatically-crosslinked gelatin hydrogels loaded with paenipeptin analogue 1 and clarithromycin exhibited potent therapeutic efficacy against carbapenem-resistant A. baumannii in murine skin wound infection.

Localized delivery of β-NGF via injectable microrods accelerates endochondral fracture repair

Currently, there are no biological approaches to accelerate bone fracture repair. Osteobiologics that promote endochondral ossification are an exciting alternative to surgically implanted bone grafts, however, the translation of osteobiologics remains elusive because of the need for localized and sustained delivery that is both safe and effective. In this regard, an injectable system composed of hydrogel-based microparticles designed to release osteobiologics in a controlled and localized manner is ideal in the context of bone fracture repair. Here, we describe poly (ethylene glycol) dimethacrylate (PEGDMA)-based microparticles, in the form of microrods, engineered to be loaded with beta nerve growth factor (β-NGF) for use in a murine tibial fracture model. In-vitro studies demonstrated that protein-loading efficiency is readily altered by varying PEGDMA macromer concentration and that β-NGF loaded onto PEGDMA microrods exhibited sustained release over a period of 7 days. In-vitro bioactivity of β-NGF was confirmed using a tyrosine receptor kinase A (Trk-A) expressing cell line, TF-1. Moreover, TF-1 cell proliferation significantly increased when incubated with β-NGF loaded PEGDMA microrods versus β-NGF in media. In-vivo studies show that PEGDMA microrods injected into the fracture calluses of mice remained in the callus for over 7 days. Importantly, a single injection of β-NGF-loaded PEGDMA microrods resulted in significantly improved fracture healing as indicated by significant increases in bone volume, trabecular connective density, and bone mineral density and a significant decrease in cartilage despite a remarkably lower dose (~111 fold) than the β-NGF in media. In conclusion, we demonstrate a novel and translational method of delivering β-NGF via injectable PEGDMA microrods to improve bone fracture repair.

259 Injectable chitosan hydrogel for localized delivery of immune checkpoint inhibitors

BackgroundSystemic delivery of checkpoint inhibitors risks the development of immune-related adverse events (irAEs) in up to 85% of patients.1 Localized delivery methods with slow-release kinetics have the potential to avoid systemic exposure and reduce irAEs. Direct tumor injection is extremely difficult, as saline-based solutions are rapidly excluded from the high-pressure tumor environment. Utilizing hydrogels as a delivery medium and local depot can address this shortcoming. To this end, we developed an injectable chitosan-based hydrogel for intratumoral delivery of checkpoint antibodies.MethodsHydrogelLow-viscosity, 80% deacetylated chitosan (Heppe Medical Chitosan; Halle, Germany) was reacted with 1-ethyl-3-(-3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) in the presence of β-glycerophosphate at room temperature for 48 hours. The mixture was then washed with ethanol and dried at 60°C. The resulting solid was dissolved in phosphate buffered saline (PBS) at concentrations from 35–70 mg/mL.In vitro release. 300 μg/mL bovine serum albumin (BSA) labeled with fluorescein-5-isothiocyanate (FITC) as a model protein drug was loaded into the hydrogel. The hydrogel was injected through a 28g needle and incubated with PBS. Samples were taken over a week period. Release kinetics were analyzed by fitting fluorescence data to zero-order, first-order, and Korsmeyer-Peppas models. To visualize retention after injection, dye-loaded hydrogels or dye in PBS alone were injected into a 0.6 wt% agar tissue phantom.In vivo imaging and tumor treatment. Flank MC38 tumors will be established in C57BL/6 mice. At tumor volumes of 50–100 mm3, 200 ug of fluorescently labeled aCTLA-4 and aPD-L1 included in the chitosan hydrogel will be delivered intratumorally. Images will be captured using an In Vivo Imaging System (IVIS). Antitumor activity will be assessed in a separate cohort using unlabeled antibodies.ResultsThe chitosan hydrogel was found to be injectable in needles as thin as 28g. After exiting the needle, the hydrogel reformed (figure 1A). Upon injection into the tissue phantom, dyed PBS immediately leaked out, primarily through the needle track, while the dyed hydrogel was retained (figure 1B). In vitro release studies demonstrated long-term, nearly zero-order, week-long sustained release (figure 1C). In vivo retention and tumor treatment studies are ongoing.Abstract 259 Figure 1Injectable chitosan hydrogel. (A) Re-formed BSA-FITC hydrogel in 1x PBS; (B) (i) Retained hydrogel in agar tissue phantom, (ii) Excluded 1x PBS in agar tissue phantom; (C) Release kinetics in 1x PBS.ConclusionsA novel injectable chitosan hydrogel was found to provide sustained release of a large model protein over a 1–2 week period with favorable in vitro kinetics. Importantly, this hydrogel can be engineered to provide faster or slower release as needed. Ongoing studies in vivo will quantify intratumoral retention, systemic dissemination, and antitumor activity.AcknowledgementsThis work is supported by the National Science Foundation Graduate Research Fellowship.ReferenceHommes J, Verheijden R, Suijkerbuijk K, Hamann D. Biomarkers of checkpoint inhibitor induced immune-related adverse events—a comprehensive review. Front Oncol 2021;10:1–16.Ethics ApprovalAnimal use was in compliance with the Public Health Service Policy on Human Care and Use of Laboratory Animals. All experiments involving laboratory animals were approved by the Institutional Animal Care and Use Committee at North Carolina State University (Protocol #19–795).

Non-Clinical In Vitro Evaluation of Antibiotic Resistance Gene-Free Plasmids Encoding Human or Murine IL-12 Intended for First-in-Human Clinical Study

Interleukin 12 (IL-12) is a key cytokine that mediates antitumor activity of immune cells. To fulfill its clinical potential, the development is focused on localized delivery systems, such as gene electrotransfer, which can provide localized delivery of IL-12 to the tumor microenvironment. Gene electrotransfer of the plasmid encoding human IL-12 is already in clinical trials in USA, demonstrating positive results in the treatment of melanoma patients. To comply with EU regulatory requirements for clinical application, which recommend the use of antibiotic resistance gene-free plasmids, we constructed and developed the production process for the clinical grade quality antibiotic resistance gene-free plasmid encoding human IL-12 (p21-hIL-12-ORT) and its ortholog encoding murine IL-12 (p21-mIL-12-ORT). To demonstrate the suitability of the p21-hIL-12-ORT or p21-mIL-12-ORT plasmid for the first-in-human clinical trial, the biological activity of the expressed transgene, its level of expression and plasmid copy number were determined in vitro in the human squamous cell carcinoma cell line FaDu and the murine colon carcinoma cell line CT26. The results of the non-clinical evaluation in vitro set the basis for further in vivo testing and evaluation of antitumor activity of therapeutic molecules in murine models as well as provide crucial data for further clinical trials of the constructed antibiotic resistance gene-free plasmid in humans.

Hyaluronate-Thiol Passivation Enhances Gold Nanoparticle Peritumoral Distribution When Administered Intratumorally in Lung Cancer

Biofouling is the unwanted adsorption of cells, proteins, or intracellular and extracellular bio-molecules that can spontaneously occur on the surface of metal nanocomplexes. It represents a major issue in bioinorganic chemistry because it leads to the creation of a protein corona, which can destabilize a colloidal solution and result in undesired macrophage-driven clearance, consequently causing failed delivery of a targeted drug-cargo. Hyaluronic acid (HA) is a bioactive, natural mucopolysaccharide with excellent antifouling properties, arising from its hydrophilic and polyanionic characteristics in physiological environments which prevent opsonization. In this study, hyaluronate-thiol (HA-SH) (MW 10 kDa) was used to surface-passivate gold nanoparticles (GNPs) synthesized using a citrate reduction method. HA functionalized GNP complexes (HA-GNPs) were characterized using absorption spectroscopy, scanning electron microscopy, zeta potential, and dynamic light scattering. GNP cellular uptake and potential dose-dependent cytotoxic effects due to treatment were evaluated in vitro in HeLa cells using ICP-OES and Trypan blue and MTT assays. Further, we quantified the in vivo biodistribution of intratumorally injected HA functionalized GNPs in Lewis Lung carcinoma (LLC) solid tumors grown on the flank of C57BL/6 mice and compared localization and retention with nascent particles. Our results reveal that HA-GNPs show overall greater peritumoral distribution (**p<0.005, 3 days post-intratumoral injection) than citrate-GNPs with reduced biodistribution in off-target organs. This property represents an advantageous step forward in localized delivery of metal nano-complexes to the infiltrative region of a tumor, which may improve the application of nanomedicine in the diagnosis and treatment of cancer.

Functional Specialization within the EXO70 Gene Family in Arabidopsis

Localized delivery of plasma-membrane and cell-wall components is a crucial process for plant cell growth. One of the regulators of secretory-vesicle targeting is the exocyst tethering complex. The exocyst mediates first interaction between transport vesicles and the target membrane before their fusion is performed by SNARE proteins. In land plants, genes encoding the EXO70 exocyst subunit underwent an extreme proliferation with 23 paralogs present in the Arabidopsis (Arabidopsis thaliana) genome. These paralogs often acquired specialized functions during evolution. Here, we analyzed functional divergence of selected EXO70 paralogs in Arabidopsis. Performing a systematic cross-complementation analysis of exo70a1 and exo70b1 mutants, we found that EXO70A1 was functionally substituted only by its closest paralog, EXO70A2. In contrast, none of the EXO70 isoforms tested were able to substitute EXO70B1, including its closest relative, EXO70B2, pointing to a unique function of this isoform. The presented results document a high degree of functional specialization within the EXO70 gene family in land plants.