Poly(ß-aminoester) Physicochemical Properties Govern the Delivery of siRNA from Electrostatically Assembled Coatings.

Localized short interfering RNA (siRNA) therapy has the potential to drive high-specificity molecular-level treatment of a variety of disease states. Unfortunately, effective siRNA therapy suffers from several barriers to its intracellular delivery. Thus, drug delivery systems that package and control the release of therapeutic siRNAs are necessary to overcome these obstacles to clinical translation. Layer-by-layer (LbL) electrostatic assembly of thin film coatings containing siRNA and protonatable, hydrolyzable poly(ß-aminoester) (PBAE) polymers is one such drug delivery strategy. However, the impact of PBAE physicochemical properties on the transfection efficacy of siRNA released from LbL thin film coatings has not been systematically characterized. In this study, we investigate the siRNA transfection efficacy of four structurally similar PBAEs in vitro. We demonstrate that small changes in structure yield large changes in physicochemical properties, such as hydrophobicity, pKa, and amine chemical structure, driving differences in the interactions between PBAEs and siRNA in polyplexes and in LbL thin film coatings for wound dressings. In our polymer set, Poly3 forms the most stable interactions with siRNA (Keff,w/w = 0.298) to slow release kinetics and enhance transfection of reporter cells in both colloidal and thin film coating approaches. This is due to its unique physiochemical properties: high hydrophobicity (clog P = 7.86), effective pKa closest to endosomal pH (pKa = 6.21), and high cooperativity in buffering (nhill = 7.2). These properties bestow Poly3 with enhanced endosomal buffering and escape properties. Taken together, this work elucidates the connections between small changes in polymer structure, emergent properties, and polyelectrolyte theory to better understand PBAE transfection efficacy.

Use of Therapeutic RNAs to Accelerate Wound Healing in Diabetic Rabbit Wounds.

Introduction: Diabetes mellitus (DM) affects over 422 million people globally. Patients with DM are subject to a myriad of complications, of which diabetic foot ulcers (DFUs) are the most common with ∼25% chance of developing these wounds throughout their lifetime. Innovation: Currently there are no therapeutic RNAs approved for use in DFUs. Use of dressings containing novel layer-by-layer (LbL)-formulated therapeutic RNAs that inhibit PHD2 and miR-210 can significantly improve diabetic wound healing. These dressings provide sustained release of therapeutic RNAs to the wounds locally without systemic side effects. Clinical Problem Addressed: Diabetic foot wounds are difficult to heal and often result in significant patient morbidity and mortality. Materials and Methods: We used the diabetic neuroischemic rabbit model of impaired wound healing. Diabetes was induced in the rabbits with alloxan, and neuroischemia was induced by ligating the central neurovascular bundle of each ear. Four 6-mm full-thickness wounds were created on each ear. A LbL technique was used to conformally coat the wound dressings with chemically modified RNAs, including an antisense oligonucleotide (antimiR) targeting microRNA-210 (miR-210), an short synthetic hairpin RNA (sshRNA) targeting PHD2, or both. Results: Wound healing was improved by the antimiR-210 but not the PHD2-sshRNA. Specific knockdown of miR-210 in tissue as measured by RT-qPCR was ∼8 Ct greater than nonspecific controls, and this apparent level of knockdown (>99%) suggests that delivery to the tissue is highly efficient at the administered dose. Discussion: Healing of ischemic/neuropathic wounds in diabetic rabbits was accelerated upon inhibition of miR-210 by LbL delivery to the wound bed. miR-210 inhibition was achieved using a chemically modified antisense RNA.

Electrostatically assembled wound dressings deliver pro-angiogenic anti-miRs preferentially to endothelial cells.

Chronic non-healing wounds occur frequently in individuals affected by diabetes, yet standard-of-care treatment leaves many patients inadequately treated or with recurring wounds. MicroRNA (miR) expression is dysregulated in diabetic wounds and drives an anti-angiogenic phenotype, but miRs can be inhibited with short, chemically-modified RNA oligonucleotides (anti-miRs). Clinical translation of anti-miRs is hindered by delivery challenges such as rapid clearance and uptake by off-target cells, requiring repeated injections, excessively large doses, and bolus dosing mismatched to the dynamics of the wound healing process. To address these limitations, we engineered electrostatically assembled wound dressings that locally release anti-miR-92a, as miR-92a is implicated in angiogenesis and wound repair. In vitro, anti-miR-92a released from these dressings was taken up by cells and inhibited its target. An in vivo cellular biodistribution study in murine diabetic wounds revealed that endothelial cells, which play a critical role in angiogenesis, exhibit higher uptake of anti-miR eluted from coated dressings than other cell types involved in the wound healing process. In a proof-of-concept efficacy study in the same wound model, anti-miR targeting anti-angiogenic miR-92a de-repressed target genes, increased gross wound closure, and induced a sex-dependent increase in vascularization. Overall, this proof-of-concept study demonstrates a facile, translational materials approach for modulating gene expression in ulcer endothelial cells to promote angiogenesis and wound healing. Furthermore, we highlight the importance of probing cellular interactions between the drug delivery system and the target cells to drive therapeutic efficacy.

pH-Responsive, Charge-Reversing Layer-by-Layer Nanoparticle Surfaces Enhance Biofilm Penetration and Eradication.

Microbes entrenched within biofilms can withstand 1000-fold higher concentrations of antibiotics, in part due to the viscous extracellular matrix that sequesters and attenuates antimicrobial activity. Nanoparticle (NP)-based therapeutics can aid in delivering higher local concentrations throughout biofilms as compared to free drugs alone, thereby enhancing the efficacy. Canonical design criteria dictate that positively charged nanoparticles can multivalently bind to anionic biofilm components and increase biofilm penetration. However, cationic particles are toxic and are rapidly cleared from circulation in vivo, limiting their use. Therefore, we sought to design pH-responsive NPs that change their surface charge from negative to positive in response to the reduced biofilm pH microenvironment. We synthesized a family of pH-dependent, hydrolyzable polymers and employed the layer-by-layer (LbL) electrostatic assembly method to fabricate biocompatible NPs with these polymers as the outermost surface. The NP charge conversion rate, dictated by polymer hydrophilicity and the side-chain structure, ranged from hours to undetectable within the experimental timeframe. LbL NPs with an increasingly fast charge conversion rate more effectively penetrated through, and accumulated throughout, wildtype (PAO1) and mutant overexpressing biomass (ΔwspF) Pseudomonas aeruginosa biofilms. Finally, tobramycin, an antibiotic known to be trapped by anionic biofilm components, was loaded into the final layer of the LbL NP. There was a 3.2-fold reduction in ΔwspF colony forming units for the fastest charge-converting NP as compared to both the slowest charge converter and free tobramycin. These studies provide a framework for the design of biofilm-penetrating NPs that respond to matrix interactions, ultimately increasing the efficacious delivery of antimicrobials.

Sustained release of BMP-2 using self-assembled layer-by-layer film-coated implants enhances bone regeneration over burst release.

Current clinical products delivering the osteogenic growth factor bone morphogenetic protein 2 (BMP-2) for bone regeneration have been plagued by safety concerns due to a high incidence of off-target effects resulting from bolus release and supraphysiological doses. Layer-by-layer (LbL) film deposition offers the opportunity to coat bone defect-relevant substrates with thin films containing proteins and other therapeutics; however, control of release kinetics is often hampered by interlayer diffusion of drugs throughout the film during assembly, which causes burst drug release. In this work, we present the design of different laponite clay diffusional barrier layer architectures in self-assembled LbL films to modulate the release kinetics of BMP-2 from the surface of a biodegradable implant. Release kinetics were tuned by incorporating laponite in different film arrangements and with varying deposition techniques to achieve release of BMP-2 over 2 days, 4 days, 14 days, and 30 days. Delivery of a low dose (0.5 µg) of BMP-2 over 2 days and 30 days using these LbL film architectures was then compared in an in vivo rat critical size calvarial defect model to determine the effect of BMP-2 release kinetics on bone regeneration. After 6 weeks, sustained release of BMP-2 over 30 days induced 3.7 times higher bone volume and 7.4 times higher bone mineral density as compared with 2-day release of BMP-2, which did not induce more bone growth than the uncoated scaffold control. These findings represent a crucial step in the understanding of how BMP-2 release kinetics influence treatment efficacy and underscore the necessity to optimize protein delivery methods in clinical formulations for bone regeneration. This work could be applied to the delivery of other therapeutic proteins for which careful tuning of the release rate is a key optimization parameter.

Massively parallel pooled screening reveals genomic determinants of nanoparticle delivery.

To accelerate the translation of cancer nanomedicine, we used an integrated genomic approach to improve our understanding of the cellular processes that govern nanoparticle trafficking. We developed a massively parallel screen that leverages barcoded, pooled cancer cell lines annotated with multiomic data to investigate cell association patterns across a nanoparticle library spanning a range of formulations with clinical potential. We identified both materials properties and cell-intrinsic features that mediate nanoparticle-cell association. Using machine learning algorithms, we constructed genomic nanoparticle trafficking networks and identified nanoparticle-specific biomarkers. We validated one such biomarker: gene expression of SLC46A3, which inversely predicts lipid-based nanoparticle uptake in vitro and in vivo. Our work establishes the power of integrated screens for nanoparticle delivery and enables the identification and utilization of biomarkers to rationally design nanoformulations.

Peptide-Based Cancer Vaccine Delivery via the STINGΔTM-cGAMP Complex.

With the advent of bioinformatic tools in efficiently predicting neo-antigens, peptide vaccines have gained tremendous attention in cancer immunotherapy. However, the delivery of peptide vaccines remains a major challenge, primarily due to ineffective transport to lymph nodes and low immunogenicity. Here, a strategy for peptide vaccine delivery is reported by first fusing the peptide to the cytosolic domain of the stimulator of interferon genes protein (STINGΔTM), then complexing the peptide-STINGΔTM protein with STING agonist 2'3' cyclic guanosine monophosphate-adenosine monophosphate (cGAMP). The process results in the formation of self-assembled cGAMP-peptide-STINGΔTM tetramers, which enables efficient lymphatic trafficking of the peptide. Moreover, the cGAMP-STINGΔTM complex acts not only as a protein carrier for the peptide, but also as a potent adjuvant capable of triggering STING signaling independent of endogenous STING protein-an especially important attribute considering that certain cancer cells epigenetically silence their endogenous STING expression. With model antigen SIINFEKL, it is demonstrated that the platform elicits effective STING signaling in vitro, draining lymph node targeting in vivo, effective T cell priming in vivo as well as antitumoral immune response in a mouse colon carcinoma model, providing a versatile solution to the challenges faced in peptide vaccine delivery.

A design approach for layer-by-layer surface-mediated siRNA delivery.

The ability to coat scaffolds and wound dressings with therapeutic short interfering RNA (siRNA) holds much potential for applications in wound healing, cancer treatment, and regenerative medicine. Layer-by-layer (LbL) technology is an effective method to formulate polyelectrolyte thin films for local delivery of siRNA; however, the formation and efficacy of LbL coatings as drug delivery systems are highly contingent on the assembly conditions. Here, we investigate the effects of LbL assembly parameters on film composition and consequent siRNA-mediated gene knockdown efficiency in vitro. Films comprising poly(ß-amino ester) (PBAE) and siRNA were built on polyglactin 910 (Vicryl) sutures consisting of poly(10% L-lactide, 90% glycolide). A fractional factorial design was employed, varying the following LbL assembly conditions: pH, ionic strength, PBAE concentration, and siRNA concentration. Effects of these parameters on PBAE loading, siRNA loading, their respective weight ratios, and in vitro siRNA-mediated knockdown were elucidated. The parameter effects were leveraged to create a rationally designed set of solution conditions that was predicted to give effective siRNA-mediated knockdown, but not included in any of the original experimental conditions. This level of knockdown with our rationally designed loading conditions (47%) is comparable to previous formulations from our lab while being simpler in construction and requiring fewer film layers, which could save time and cost in manufacturing. This study highlights the importance of LbL solution conditions in the preparation of surface-mediated siRNA delivery systems and presents an adaptable methodology for extending these electrostatically-assembled coatings to the delivery of other therapeutic nucleic acids. STATEMENT OF SIGNIFICANCE: Short interfering RNA (siRNA) therapeutics are powerful tools to silence aberrant gene expression in the diseased state; however, the clinical utility of these therapies relies on effective controlled delivery approaches. Electrostatic self-assembly through the layer-by-layer (LbL) process enables direct siRNA release from surfaces, but this method is highly dependent upon the specific solution conditions used. Here, we use a fractional factorial design to illustrate how these assembly conditions impact composition of siRNA-eluting LbL thin films. We then elucidate how these properties mediate in vitro transfection efficacy. Ultimately, this work presents a significant step towards understanding how optimization of assembly conditions for surface-mediated LbL delivery can promote transfection efficacy while reducing the processing and material required.

Oxidation-Responsive, Tunable Growth Factor Delivery from Polyelectrolyte-Coated Implants.

Polyelectrolyte multilayer (PEM) coatings, constructed on the surfaces of tissue engineering scaffolds using layer-by-layer assembly (LbL), promote sustained release of therapeutic molecules and have enabled regeneration of large-scale, pre-clinical bone defects. However, these systems primarily rely on non-specific hydrolysis of PEM components to foster drug release, and their pre-determined drug delivery schedules potentially limit future translation into innately heterogeneous patient populations. To trigger therapeutic delivery directly in response to local environmental stimuli, an LbL-compatible polycation solely degraded by cell-generated reactive oxygen species (ROS) was synthesized. These thioketal-based polymers were selectively cleaved by physiologic doses of ROS, stably incorporated into PEM films alongside growth factors, and facilitated tunable release of therapeutic bone morphogenetic protein-2 (BMP-2) upon oxidation. These coatings' sensitivity to oxidation was also dependent on the polyanions used in film construction, providing a simple method for enhancing ROS-mediated protein delivery in vitro. Correspondingly, when implanted in critically-sized rat calvarial defects, the most sensitive ROS-responsive coatings generated a 50% increase in bone regeneration compared with less sensitive formulations and demonstrated a nearly threefold extension in BMP-2 delivery half-life over conventional hydrolytically-sensitive coatings. These combined results highlight the potential of environmentally-responsive PEM coatings as tunable drug delivery systems for regenerative medicine.

Approaches to Modulate the Chronic Wound Environment Using Localized Nucleic Acid Delivery.

Significance: Nonhealing wounds have been the subject of decades of basic and clinical research. Despite new knowledge about the biology of impaired wound healing, little progress has been made in treating chronic wounds, leaving patients with few therapeutic options. Diabetic ulcers are a particularly common form of nonhealing wound. Recent Advances: Recently, investigation of therapeutic nucleic acids (TNAs), including plasmid DNA, small interfering RNA, microRNA mimics, anti-microRNA oligonucleotides, messenger RNA, and antisense oligonucleotides, has created a new treatment strategy for chronic wounds. TNAs can modulate the wound toward a prohealing environment by targeting gene pathways associated with inflammation, proteases, cell motility, angiogenesis, epithelialization, and oxidative stress. A variety of delivery systems have been investigated for TNAs, including dendrimers, lipid nanoparticles (NPs), polymeric micelles, polyplexes, metal NPs, and hydrogels. This review summarizes recent developments in TNA delivery for therapeutic targets associated with chronic wounds, with an emphasis on diabetic ulcers. Critical Issues: Translational potential of TNAs remains a key challenge; we highlight some drug delivery approaches for TNAs that may hold promise. We also describe current commercial efforts to locally deliver nucleic acids to modulate the wound environment. Future Directions: Localized nucleic acid delivery holds promise for the treatment of nonhealing chronic wounds. Future efforts to improve targeting of these nucleic acid therapies in the wound with both spatial and temporal control through drug delivery systems will be crucial to successful clinical translation.

Re-engaging students in medical school accreditation: the independent student analysis action (ISA2) approach.

This article was migrated. The article was marked as recommended. Background: Student satisfaction with key aspects of a medical school program plays a major role in an institution's accreditation in the United States (US). There is limited evidence regarding how to best engage students in responding to areas of lower student satisfaction that are identified through the national Liaison Committee on Medication Education (LCME) accreditation self-study process. Methods:We present a student-led innovation to promote greater levels of student engagement throughout Harvard Medical School's re-accreditation experience, which we refer to as the Independent Student Analysis Action (ISA 2) process. This innovation built directly upon the Independent Student Analysis (ISA) survey, which is expected by the LCME for accreditation. The ISA 2 process allowed medical student leaders to leverage ISA results to identify 11 priority areas that had relatively lower levels of satisfaction and subsequently coordinate focused, time-limited ISA 2 working groups to address these problematic areas. These working groups then presented their solutions to the student body, and a follow-up survey gauged satisfaction with these areas in light of the changes made. Results: The ISA 2 process engaged over 110 students, faculty, and staff. The majority of the student body completed the follow-up survey, which demonstrated higher levels of satisfaction with these previously problematic areas as identified in the original ISA survey. Further, 96% of students reported being satisfied with the ISA 2 process as a mechanism for utilizing student feedback in the ISA to create meaningful institutional changes. Conclusions: The ISA 2 served as a powerful convening mechanism for engaging a large number of students in our institution's re-accreditation efforts. Other medical schools looking to involve students in their continuous quality improvement systems and accreditation experience may benefit from reviewing and customizing this model to their institution's needs.

Clickotine, A Personalized Smartphone App for Smoking Cessation: Initial Evaluation.

BACKGROUND: Tobacco smoking is the leading cause of preventable death in the United States, and the annual economic burden attributable to smoking exceeds US $300 billion. Obstacles to smoking cessation include limited access and adherence to effective cessation interventions. Technology can help overcome these obstacles; many smartphone apps have been developed to aid smoking cessation, but few that conform to the US clinical practice guideline (USCPG) have been rigorously tested and reported in the literature. Clickotine is a novel smartphone app for smoking cessation, designed to deliver the essential features of the USCPG and engineered to engage smokers by personalizing intervention components. OBJECTIVE: Our objective was to assess the engagement, efficacy, and safety of Clickotine in an initial, single-arm study. Outcomes measured were indicators of engagement with the smartphone app (number of app opens, number of interactions with the Clickotine program, and weeks active with Clickotine), cessation outcomes of 7- and 30-day self-reported abstinence from smoking, and negative health events. METHODS: We recruited US residents between 18 and 65 years of age who owned an iPhone and smoked 5 or more cigarettes daily for the study via online advertising. Respondents were prescreened for eligibility by telephone and, if appropriate, directed to a Web portal to provide informed consent, confirm eligibility, and download the Clickotine app. Participants completed study assessments via the online portal at baseline and after 8 weeks. Data were collected in Amazon S3 with no manual data entry, and access to all data was maximally restrictive, logged, and auditable. RESULTS: A total of 416 participants downloaded the app and constituted the intention-to-treat (ITT) sample. On average, participants opened the Clickotine app 100.6 times during the 8-week study (median 69), logged 214.4 interactions with the Clickotine program (median 178), and remained engaged with Clickotine for 5.3 weeks (median 5). Among the ITT sample, 45.2% (188/416) reported 7-day abstinence and 26.2% (109/416) reported 30-day abstinence from smoking after 8 weeks. Completer analysis focused on 365 (87.7%) of the 416 enrolled participants who completed the 8-week questionnaire revealed that 51.5% (188/365) of completers reported 7-day abstinence and 29.9% (109/365) reported 30-day abstinence. Few adverse events, mostly consistent with nicotine withdrawal symptoms, were reported and overall no safety signal was detected. CONCLUSIONS: In this initial single-arm trial, Clickotine users appeared to demonstrate encouraging indicators of engagement in terms of the number of app opens, number of program interactions, and continued engagement over time. Clickotine users reported encouraging quit rates while reporting few adverse events. Future research is warranted to assess Clickotine's efficacy in a randomized controlled trial. TRIAL REGISTRATION: Clinicaltrials.gov NCT02656745; https://clinicaltrials.gov/ct2/show/NCT02656745 (Archived by WebCite at http://www.webcitation.org/6peTT4x60).

Vertical-flow paper SERS system for therapeutic drug monitoring of flucytosine in serum.

A number of life-saving drugs require therapeutic drug monitoring (TDM) for safe and effective use. Currently, however, TDM is performed using sophisticated analytical techniques relegated to central labs, increasing the cost per test and time to answer. Here, using a novel vertical flow membrane system with inkjet-printed surface enhanced Raman sensors, along with a portable spectrometer, we demonstrate a low cost and easy to use device to quantify levels of flucytosine, an antifungal that requires TDM for effective patient care, from undiluted human serum. To our knowledge, this work represents the first report of a passive vertical flow sample cleanup method with surface enhanced Raman detection. We first investigated and optimized the parameters of the vertical flow system for the detection of flucytosine in spiked serum samples. Then, using an optimized vertical-flow system utilizing nitrocellulose membranes and a paper SERS sensor, we achieved detection of down to 10 µg mL-1 flucytosine in undiluted serum, with quantitative detection across the entire therapeutic range. This system reduces the assay time to about 15 min, far quicker than the current gold standards. We anticipate that this novel system will enable near-patient therapeutic drug monitoring, leading to the safe and effective administration of a number of life-saving drugs. Furthermore, it will spawn the development of SERS detection systems capable of separating target analytes from real-world biological matrices.