A novel mechanism for the loss of mRNA activity in lipid nanoparticle delivery systems.

Lipid nanoparticle (LNP)-formulated mRNA vaccines were rapidly developed and deployed in response to the SARS-CoV-2 pandemic. Due to the labile nature of mRNA, identifying impurities that could affect product stability and efficacy is crucial to the long-term use of nucleic-acid based medicines. Herein, reversed-phase ion pair high performance liquid chromatography (RP-IP HPLC) was used to identify a class of impurity formed through lipid:mRNA reactions; such reactions are typically undetectable by traditional mRNA purity analytical techniques. The identified modifications render the mRNA untranslatable, leading to loss of protein expression. Specifically, electrophilic impurities derived from the ionizable cationic lipid component are shown to be responsible. Mechanisms implicated in the formation of reactive species include oxidation and subsequent hydrolysis of the tertiary amine. It thus remains critical to ensure robust analytical methods and stringent manufacturing control to ensure mRNA stability and high activity in LNP delivery systems.

Erythropoietin inhalation enhances adult canine alveolar-capillary formation following pneumonectomy.

Paracrine erythropoietin (EPO) signaling in the lung recruits endothelial progenitor cells, promotes cell maturation and angiogenesis, and is upregulated during canine postpneumonectomy (PNX) compensatory lung growth. To determine whether inhalational delivery of exogenous EPO augments endogenous post-PNX lung growth, adult canines underwent right PNX and received, via a permanent tracheal stoma, weekly nebulization of recombinant human EPO-containing nanoparticles or empty nanoparticles (control) for 16 wk. Lung function was assessed under anesthesia pre- and post-PNX. The remaining lobes were fixed for detailed morphometric analysis. Compared with control treatment, EPO delivery significantly increased serum EPO concentration without altering systemic hematocrit or hemoglobin concentration and abrogated post-PNX lipid oxidative stress damage. EPO delivery modestly increased post-PNX volume densities of the alveolar septum per unit of lung volume and type II epithelium and endothelium per unit of septal tissue volume in selected lobes. EPO delivery also augmented the post-PNX increase in alveolar double-capillary profiles, a marker of intussusceptive capillary formation, in all remaining lobes. EPO treatment did not significantly alter absolute resting lung volumes, lung and membrane diffusing capacities, alveolar-capillary blood volume, pulmonary blood flow, lung compliance, or extravascular alveolar tissue volumes or surface areas. Results established the feasibility of chronic inhalational delivery of growth-modifying biologics in a large animal model. Exogenous EPO selectively enhanced cytoprotection and alveolar angiogenesis in remaining lobes but not whole-lung extravascular tissue growth or resting function; the nonuniform response contributes to structure-function discrepancy, a major challenge for interventions aimed at amplifying the innate potential for compensatory lung growth.

Highly photostable nanogels for fluorescence-based theranostics.

A novel photo-crosslinkable nanogel is prepared from a biodegradable polymer template with intrinsic photoluminescence and high photostability. The fluorescent nanogels display excellent biodegradability and cytocompatibility owed to the facile synthesis scheme involving a solvent-and surfactant-free one-pot reaction, derived entirely from biocompatible monomers citric acid, maleic acid, L-cysteine, and poly(ethylene glycol). The resultant nanogels are less than 200 nm in diameter with a narrow size distribution and monodispersity, and demonstrate long-term structural stability in biological buffer for two weeks. To gauge potential in theranostic applications, the fluorescent nanogels were surface functionalized with biologically active RGD peptides and encapsulated with active anti-cancer drug Doxorubicin, resulting in a pH-responsive controlled drug release in acidic pH resembling tumor environments. The strong fluorescence of the nanogels enabled tracking of targeted drug delivery, showing that drug-loaded nanogels homed into the cytoplasmic regions of prostate cancer cells to significantly induce cell death. These photo-crosslinkable and biodegradable nanogels pose as a strong candidate for theranostic medicine, demonstrating versatile functionalization, high stability in biological buffers, and capacity for real-time fluorescence-based monitoring of targeted drug delivery.

Perfusion-related stimuli for compensatory lung growth following pneumonectomy.

Following pneumonectomy (PNX), two separate mechanical forces act on the remaining lung: parenchymal stress caused by lung expansion, and microvascular distension and shear caused by increased perfusion. We previously showed that parenchymal stress and strain explain approximately one-half of overall compensation; the remainder was presumptively attributed to perfusion-related factors. In this study, we directly tested the hypothesis that perturbation of regional pulmonary perfusion modulates post-PNX lung growth. Adult canines underwent banding of the pulmonary artery (PAB) to the left caudal (LCa) lobe, which caused a reduction in basal perfusion to LCa lobe without preventing the subsequent increase in its perfusion following right PNX while simultaneously exaggerating the post-PNX increase in perfusion to the unbanded lobes, thereby creating differential perfusion changes between banded and unbanded lobes. Control animals underwent sham pulmonary artery banding followed by right PNX. Pulmonary function, regional pulmonary perfusion, and high-resolution computed tomography of the chest were analyzed pre-PNX and 3-mo post-PNX. Terminally, the remaining lobes were fixed for detailed morphometric analysis. Results were compared with corresponding lobes in two control (Sham banding and normal unoperated) groups. PAB impaired the indices of post-PNX extravascular alveolar tissue growth by up to 50% in all remaining lobes. PAB enhanced the expected post-PNX increase in alveolar capillary formation, measured by the prevalence of double-capillary profiles, in both unbanded and banded lobes. We conclude that perfusion distribution provides major stimuli for post-PNX compensatory lung growth independent of the stimuli provided by lung expansion and parenchymal stress and strain.

Nanoparticle facilitated inhalational delivery of erythropoietin receptor cDNA protects against hyperoxic lung injury.

Our goals were to develop and establish nanoparticle (NP)-facilitated inhalational gene delivery, and to validate its biomedical application by testing the hypothesis that targeted upregulation of pulmonary erythropoietin receptor (EpoR) expression protects against lung injury. Poly-lactic-co-glycolic acid (PLGA) NPs encapsulating various tracers were characterized and nebulizated into rat lungs. Widespread NP uptake and distribution within alveolar cells were visualized by magnetic resonance imaging, and fluorescent and electron microscopy. Inhalation of nebulized NPs bearing EpoR cDNA upregulated pulmonary EpoR expression and downstream signal transduction (ERK1/2 and STAT5 phosphorylation) in rats for up to 21 days, and attenuated hyperoxia-induced damage in lung tissue based on apoptosis, oxidative damage of DNA, protein and lipid, tissue edema, and alveolar morphology compared to vector-treated control animals. These results establish the feasibility and therapeutic efficacy of NP-facilitated cDNA delivery to the lung, and demonstrate that targeted pulmonary EpoR upregulation mitigates acute oxidative lung damage. FROM THE CLINICAL EDITOR: Acute lung injury often results in significant morbidity and mortality, and current therapeutic modalities have proven to be ineffective. In this article, the authors developed nanocarrier based gene therapy in an attempt to upregulate the expression of pulmonary erythropoietin receptor in an animal model. Inhalation delivery resulted in reduction of lung damage.

Design of antimicrobial peptides conjugated biodegradable citric acid derived hydrogels for wound healing.

Wound healing is usually facilitated by the use of a wound dressing that can be easily applied to cover the wound bed, maintain moisture, and avoid bacterial infection. In order to meet all of these requirements, we developed an in situ forming biodegradable hydrogel (iFBH) system composed of a newly developed combination of biodegradable poly(ethylene glycol) maleate citrate (PEGMC) and poly(ethylene glycol) diacrylate (PEGDA). The in situ forming hydrogel systems are able to conform to the wound shape in order to cover the wound completely and prevent bacterial invasion. A 2(k) factorial analysis was performed to examine the effects of polymer composition on specific properties, including the curing time, Young's modulus, swelling ratio, and degradation rate. An optimized iFBH formulation was achieved from the systematic factorial analysis. Further, in vitro biocompatibility studies using adult human dermal fibroblasts (HDFs) confirmed that the hydrogels and degradation products are not cytotoxic. The iFBH wound dressing was conjugated and functionalized with antimicrobial peptides as well. Evaluation against bacteria both in vitro and in vivo in rats demonstrated that the peptide-incorporated iFBH wound dressing offered excellent bacteria inhibition and promoted wound healing. These studies indicated that our in situ forming antimicrobial biodegradable hydrogel system is a promising candidate for wound treatment.

Polymeric nanoparticles for pulmonary protein and DNA delivery.

Polymeric nanoparticles (NPs) are promising carriers of biological agents to the lung due to advantages including biocompatibility, ease of surface modification, localized action and reduced systemic toxicity. However, there have been no studies extensively characterizing and comparing the behavior of polymeric NPs for pulmonary protein/DNA delivery both in vitro and in vitro. We screened six polymeric NPs: gelatin, chitosan, alginate, poly(lactic-co-glycolic) acid (PLGA), PLGA-chitosan and PLGA-poly(ethylene glycol) (PEG), for inhalational protein/DNA delivery. All NPs except PLGA-PEG and alginate were <300nm in size with a bi-phasic core compound release profile. Gelatin, PLGA NPs and PLGA-PEG NPs remained stable in deionized water, serum, saline and simulated lung fluid (Gamble's solution) over 5days. PLGA-based NPs and natural polymer NPs exhibited the highest cytocompatibility and dose-dependent in vitro uptake, respectively, by human alveolar type-1 epithelial cells. Based on these profiles, gelatin and PLGA NPs were used to encapsulate plasmid DNA encoding yellow fluorescent protein (YFP) or rhodamine-conjugated erythropoietin (EPO) for inhalational delivery to rats. Following a single inhalation, widespread pulmonary EPO distribution persisted for up to 10days while increasing YFP expression was observed for at least 7days for both NPs. The overall results support both PLGA and gelatin NPs as promising carriers for pulmonary protein/DNA delivery.

Fluorescence imaging enabled biodegradable photostable polymeric micelles.

Amphiphilic biodegradable photoluminescent polymers (ABPLPs) composed of a biodegradable fluorescent polymer and methoxy poly (ethyleneglycol) demonstrate intrinsic bright, tunable, and stable fluorescence emission. ABPLP micelles elicit minor cellular toxicity and can be used for cell and tissue imaging both in vitro and in vivo.

Citrate-based Biodegradable Injectable hydrogel Composites for Orthopedic Applications.

Previous studies have confirmed that natural bone apatite crystals are bound with citrate-rich molecules. Citrates on apatite crystals impact bone development and its load-bearing function. However, such understanding has never been translated into bone biomaterials design. Herein, a first citrate-based injectable composite material for orthopedic applications is developed based on our recently developed biodegradable poly(ethylene glycol) maleate citrate (PEGMC) and hydroxyapatite (HA). PEGMC contains rich carboxylic groups that could chelate with calcium-containing HA thus facilitating polymer/HA interactions, similar to natural citrate-bound apatite crystal. The crosslinking of poly(ethylene glycol) diacrylate (PEGDA) with PEGMC/HA composites allows an addition control over degradation and mechanical properties of the crosslinked PEGMC/HA (CPEGMC/HA) composites. CPEGMC/HA composite can serve as an ideal injectable cell carrier as confirmed by the enhanced DNA content, ALP activity, and calcium production through a human fetal osteoblast encapsulation study. Ex vivo study on porcine femoral head demonstrated that PEGMC/HA is a potentially promising injectable biodegradable bone material for the treatment of osteonecrosis of the femoral head. Development of biodegradable citrate-based injectable PEGMC/HA composite is an initial step for the development of the next generation of bone tissue engineering and orthopedic biomaterials.

Injectable citrate-based mussel-inspired tissue bioadhesives with high wet strength for sutureless wound closure.

The existing surgical adhesives are not ideal for wet tissue adhesion required in many surgeries such as those for internal organs. Developing surgical adhesives with strong wet tissue adhesion, controlled degradability and mechanical properties, and excellent biocompatibility has been a significant challenge. Herein, learning from nature, we report a one-step synthesis of a family of injectable citrate-based mussel-inspired bioadhesives (iCMBAs) for surgical use. Within the formulations investigated, iCMBAs showed 2.5-8.0 folds stronger wet tissue adhesion strength over the clinically used fibrin glue, demonstrated controlled degradability and tissue-like elastomeric mechanical properties, and exhibited excellent cyto/tissue-compatibility both in vitro and in vivo. iCMBAs were able to stop bleeding instantly and suturelessly, and close wounds (2 cm long×0.5 cm deep) created on the back of Sprague-Dawley rats, which is impossible when using existing gold standard, fibrin glue, due to its weak wet tissue adhesion strength. Equally important, the new bioadhesives facilitate wound healing, and are completely degraded and absorbed without eliciting significant inflammatory response. Our results support that iCMBA technology is highly translational and could have broad impact on surgeries where surgical tissue adhesives, sealants, and hemostatic agents are used.

A Rheological Study of Biodegradable Injectable PEGMC/HA Composite Scaffolds.

Injectable biodegradable hydrogels, which can be delivered in a minimally invasive manner and formed in situ, have found a number of applications in pharmaceutical and biomedical applications, such as drug delivery and tissue engineering. We have recently developed an in situ crosslinkable citric acid-based biodegradable poly (ethylene glycol) maleate citrate (PEGMC)/hydroxyapatite (HA) composite, which shows promise for use in bone tissue engineering. In this study, the mechanical properties of the PEGMC/HA composites were studied in dynamic linear rheology experiments. Critical parameters such as monomer ratio, crosslinker, initiator, and HA concentrations were varied to reveal their effect on the extent of crosslinking as they control the mechanical properties of the resultant gels. The rheological studies, for the first time, allowed us investigating the physical interactions between HA and citric acid-based PEGMC. Understanding the viscoelastic properties of the injectable gel composites is crucial in formulating suitable injectable PEGMC/HA scaffolds for bone tissue engineering, and should also promote the other biomedical applications based on citric acid-based biodegradable polymers.

Development of Photocrosslinkable Urethane-Doped Polyester Elastomers for Soft Tissue Engineering.

Finding an ideal biomaterial with the proper mechanical properties and biocompatibility has been of intense focus in the field of soft tissue engineering. This paper reports on the synthesis and characterization of a novel crosslinked urethane-doped polyester elastomer (CUPOMC), which was synthesized by reacting a previously developed photocrosslinkable poly (octamethylene maleate citrate) (POMC) prepolymers (pre-POMC) with 1,6-hexamethylene diisocyanate (HDI) followed by thermo- or photo-crosslinking polymerization. The mechanical properties of the CUPOMCs can be tuned by controlling the molar ratios of pre-POMC monomers, and the ratio between the prepolymer and HDI. CUPOMCs can be crosslinked into a 3D network through polycondensation or free radical polymerization reactions. The tensile strength and elongation at break of CUPOMC synthesized under the known conditions range from 0.73±0.12MPa to 10.91±0.64MPa and from 72.91±9.09% to 300.41±21.99% respectively. Preliminary biocompatibility tests demonstrated that CUPOMCs support cell adhesion and proliferation. Unlike the pre-polymers of other crosslinked elastomers, CUPOMC pre-polymers possess great processability demonstrated by scaffold fabrication via a thermally induced phase separation method. The dual crosslinking methods for CUPOMC pre-polymers should enhance the versatile processability of the CUPOMC used in various conditions. Development of CUPOMC should expand the choices of available biodegradable elastomers for various biomedical applications such as soft tissue engineering.

Scaffold Sheet Design Strategy for Soft Tissue Engineering.

Creating heterogeneous tissue constructs with an even cell distribution and robust mechanical strength remain important challenges to the success of in vivo tissue engineering. To address these issues, we are developing a scaffold sheet tissue engineering strategy consisting of thin (∼200 µm), strong, elastic, and porous crosslinked urethane-doped polyester (CUPE) scaffold sheets that are bonded together chemically or through cell culture. Suture retention of the tissue constructs (four sheets) fabricated by the scaffold sheet tissue engineering strategy is close to the surgical requirement (1.8 N) rendering their potential for immediate implantation without a need for long cell culture times. Cell culture results using 3T3 fibroblasts show that the scaffold sheets are bonded into a tissue construct via the extracellular matrix produced by the cells after 2 weeks of in vitro cell culture.

Citric acid-derived in situ crosslinkable biodegradable polymers for cell delivery.

Herein, we report a first citric acid (CA)-derived in situ crosslinkable biodegradable polymer, poly(ethylene glycol) maleate citrate (PEGMC). The synthesis of PEGMC could be carried out via a one-pot polycondensation reaction without using organic solvents or catalysts. PEGMC could be in situ crosslinked into elastomeric PPEGMC hydrogels. The performance of hydrogels in terms of swelling, degradation, and mechanical properties were highly dependent on the molar ratio of monomers, crosslinker concentration, and crosslinking mechanism used in the synthesis process. Cyclic conditioning tests showed that PPEGMC hydrogels could be compressed up to 75% strain without permanent deformation and with negligible hysteresis. Water-soluble PEGMC demonstrated excellent cytocompatibilty in vitro. The degradation products of PPEGMC also showed minimal cytotoxicity in vitro. Animal studies in rats clearly demonstrated the excellent injectability of PEGMC and degradability of the in situ-formed PPEGMC. PPEGMC elicited minimal inflammation in the early stages post-injection and was completely degraded within 30 days in rats. In conclusion, the development of CA-derived injectable biodegradable PEGMC presents numerous opportunities for material innovation and offers excellent candidate materials for in situ tissue engineering and drug delivery applications.

Citric-acid-derived photo-cross-linked biodegradable elastomers.

Citric-acid-derived thermally cross-linked biodegradable elastomers (CABEs) have recently received significant attention in various biomedical applications, including tissue-engineering orthopedic devices, bioimaging and implant coatings. However, citric-acid-derived photo-cross-linked biodegradable elastomers are rarely reported. Herein, we report a novel photo-cross-linked biodegradable elastomer, referred to as poly(octamethylene maleate citrate) (POMC), which preserves pendant hydroxyl and carboxylic functionalities after cross-linking for the potential conjugation of biologically active molecules. Pre-POMC is a low-molecular-mass pre-polymer with an average molecular mass between 701 and 1291 Da. POMC networks are soft and elastic with an initial modulus of 0.07 to 1.3 MPa and an elongation-at-break between 38 and 382%. FT-IR-ATR results confirmed the successful surface immobilization of type-I collagen onto POMC films, which enhanced in vitro cellular attachment and proliferation. Photo-polymerized POMC films implanted subcutaneously into Sprague-Dawley rats demonstrated minimal in vivo inflammatory responses. The development of POMC enriches the family of citric-acid-derived biodegradable elastomers and expands the available biodegradable polymers for versatile needs in biomedical applications.

Synthesis and characterization of a biodegradable elastomer featuring a dual crosslinking mechanism.

The need for advanced materials in emerging technologies such as tissue engineering has prompted increased research to produce novel biodegradable polymers elastic in nature and mechanically compliant with the host tissue. We have developed a soft biodegradable elastomeric platform biomaterial created from citric acid, maleic anhydride, and 1,8-octanediol, poly(octamethylene maleate (anhydride) citrate) (POMaC), which is able to closely mimic the mechanical properties of a wide range of soft biological tissues. POMaC features a dual crosslinking mechanism, which allows for the option of the crosslinking POMaC using UV irradiation and/or polycondensation to fit the needs of the intended application. The material properties, degradation profiles, and functionalities of POMaC thermoset networks can all be tuned through the monomer ratios and the dual crosslinking mechanism. POMaC polymers displayed an initial modulus between 0.03 and 1.54 MPa, and elongation at break between 48% and 534% strain. In vitro and in vivo evaluation using cell culture and subcutaneous implantation, respectively, confirmed cell and tissue biocompatibility. POMaC biodegradable polymers can also be combined with MEMS technology to fabricate soft and elastic 3D microchanneled scaffolds for tissue engineering applications. The introduction of POMaC will expand the choices of available biodegradable polymeric elastomers. The dual crosslinking mechanism for biodegradable elastomer design should contribute to biomaterials science.