Development of mRNA Lipid Nanoparticles: Targeting and Therapeutic Aspects.

Lipid nanoparticles (LNPs) have emerged as leading non-viral carriers for messenger RNA (mRNA) delivery in clinical applications. Overcoming challenges in safe and effective mRNA delivery to target tissues and cells, along with controlling release from the delivery vehicle, remains pivotal in mRNA-based therapies. This review elucidates the structure of LNPs, the mechanism for mRNA delivery, and the targeted delivery of LNPs to various cells and tissues, including leukocytes, T-cells, dendritic cells, Kupffer cells, hepatic endothelial cells, and hepatic and extrahepatic tissues. Here, we discuss the applications of mRNA-LNP vaccines for the prevention of infectious diseases and for the treatment of cancer and various genetic diseases. Although challenges remain in terms of delivery efficiency, specific tissue targeting, toxicity, and storage stability, mRNA-LNP technology holds extensive potential for the treatment of diseases.

Construction of Smart Biomaterials for Promoting Diabetic Wound Healing.

Diabetes mellitus is a complicated metabolic disease that has become one of the fastest-growing health crises in modern society. Diabetic patients may suffer from various complications, and diabetic foot is one of them. It can lead to increased rates of lower-extremity amputation and mortality, even seriously threatening the life and health of patients. Because its healing process is affected by various factors, its management and treatment are very challenging. To address these problems, smart biomaterials have been developed to expedite diabetic wound closure and improve treatment outcomes. This review begins with a discussion of the basic mechanisms of wound recovery and the limitations of current dressings used for diabetic wound healing. Then, the categories and characteristics of the smart biomaterial scaffolds, which can be utilized as a delivery system for drugs with anti-inflammatory activity, bioactive agency, and antibacterial nanoparticles for diabetic wound treatment were described. In addition, it can act as a responsive system to the stimulus of the pH, reactive oxygen species, and glucose concentration from the wound microenvironment. These results show that smart biomaterials have an enormous perspective for the treatment of diabetic wounds in all stages of healing. Finally, the advantages of the construction of smart biomaterials are summarized, and possible new strategies for the clinical management of diabetic wounds are proposed.

Integrin-Targeted, Short Interfering RNA Nanocomplexes for Neuroblastoma Tumor-Specific Delivery Achieve MYCN Silencing with Improved Survival.

The authors aim to develop siRNA therapeutics for cancer that can be administered systemically to target tumors and retard their growth. The efficacy of systemic delivery of siRNA to tumors with nanoparticles based on lipids or polymers is often compromised by their rapid clearance from the circulation by the liver. Here, multifunctional cationic and anionic siRNA nanoparticle formulations are described, termed receptor-targeted nanocomplexes (RTNs), that comprise peptides for siRNA packaging into nanoparticles and receptor-mediated cell uptake, together with lipids that confer nanoparticles with stealth properties to enhance stability in the circulation, and fusogenic properties to enhance endosomal release within the cell. Intravenous administration of RTNs in mice leads to predominant accumulation in xenograft tumors, with very little detected in the liver, lung, or spleen. Although non-targeted RTNs also enter the tumor, cell uptake appears to be RGD peptide-dependent indicating integrin-mediated uptake. RTNs with siRNA against MYCN (a member of the Myc family of transcription factors) in mice with MYCN-amplified neuroblastoma tumors show significant retardation of xenograft tumor growth and enhanced survival. This study shows that RTN formulations can achieve specific tumor-targeting, with minimal clearance by the liver and so enable delivery of tumor-targeted siRNA therapeutics.

Overcoming Nanoparticle-Mediated Complement Activation by Surface PEG Pairing.

Many PEGylated nanoparticles activate the complement system, which is an integral component of innate immunity. This is of concern as uncontrolled complement activation is potentially detrimental and contributes to disease pathogenesis. Here, it is demonstrated that, in contrast to carboxyPEG2000-stabilized poly(lactic-co-glycolic acid) nanoparticles, surface camouflaging with appropriate combinations and proportions of carboxyPEG2000 and methoxyPEG550 can largely suppress nanoparticle-mediated complement activation through the lectin pathway. This is attributed to the ability of the short, rigid methoxyPEG550 chains to laterally compress carboxyPEG2000 molecules to become more stretched and assume an extended, random coil configuration. As supported by coarse-grained molecular dynamics simulations, these conformational attributes minimize statistical protein binding/intercalation, thereby affecting sequential dynamic processes in complement convertase assembly. Furthermore, PEG pairing has no additional effect on nanoparticle longevity in the blood and macrophage uptake. PEG pairing significantly overcomes nanoparticle-mediated complement activation without the need for surface functionalization with complement inhibitors.

Engineering Pseudomonas entomophila for synthesis of copolymers with defined fractions of 3-hydroxybutyrate and medium-chain-length 3-hydroxyalkanoates.

Polyhydroxyalkanoates (PHA) composed of both short-chain-length (SCL) and medium-chain-length (MCL) monomers (SCL-co-MCL PHA) combine the advantages of high strength and elasticity provided by SCL PHA and MCL PHA, respectively. Synthesis of SCL-co-MCL PHA, namely, copolymers of 3-hydroxybutyrate (3HB) and MCL 3-hydroxyalkanoates (3HA) such as 3-hydroxydecanoate (3HD) and longer chain 3HA, has been a challenge for a long time. This study aims to engineer Pseudomonas entomophila for synthesizing P(3HB-co-MCL 3HA) via weakening its ß-oxidation pathway combined with insertion of 3HB synthesis pathway consisting of ß-ketothiolase (phaA) and acetoacetyl-CoA reductase (phaB). 3HB and MCL 3HA polymerization is catalyzed by a low specificity PHA synthase (phaC), namely, mutated PhaC61-3. The link between the fatty acid de novo synthesis and PHA synthesis was further blocked to increase the supply for SCL and MCL monomers in P. entomophila. The so-constructed P. entomophila was successfully used to synthesize novel PHA copolymers of P(3HB-co-3HD), P(3HB-co-3HDD) and P(3HB-co-3H9D) consisting of 3HB and 3-hydroxydecanoate (3HD), 3-hydroxydodecanoate (3HDD) and 3-hydroxy-9-decanent (3H9D), respectively. MCL 3HA compositions of P(3HB-co-3HD) and P(3HB-co-3HDD) can be adjusted from 0 to approximate 100 mol%. Results demonstrated that the engineered P. entomophila could be a platform for tailor-made P(3HB-co-MCL 3HA).

Synthesis and Characterization of Electroconductive PHA- graft-Graphene Nanocomposites.

With increasing demand of environmentally friendly materials, development on biobased polymers such as polyhydroxyalkanoate (PHA) is indispensable. An unsaturated PHA, namely, poly(3-hydroxydodecanoate- co-3-hydroxy-9-decenoate), short as P(3HDD- co-3H9D), provides possibilities for functionalization. Two different strategies are explored for synthesis of PHA- graft-graphene nanocomposites with graphene content ranging from 0.2 to 1.5 wt %. Chemical structures of intermediates and products were confirmed by Fourier transform infrared spectroscopy (FTIR) and nuclear magnetic resonance (NMR). Uniform dispersion of graphene was observed in formed PHA nanocomposites under scanning electron microscopy (SEM), transmission electron microscopy (TEM), and atomic force microscopy (AFM). PHA- graft-graphene nanocomposites exhibited higher thermal degradation temperature and enhanced electricity conductivity compared with that of neat PHA. Moreover, lower critical filling content and lower electrical resistivity at same graphene content demonstrated enhanced electrical conductivity of PHA- graft-graphene nanocomposites compared with previously reported blends. The lowest electrical resistivity was 2 Ω·m in sample PHA- graft-graphene nanocomposites with approximately 1.5 wt % graphene content.

Correction to: Environmental biodegradation of haloarchaea-produced poly(3-hydroxybutyrate-co-3-hydroxyvalerate) in activated sludge.

The published online version contains mistake in the funding information. Instead of 30370096, it should have been 31370096.

Synthesis, Characterization and Application of Thermoresponsive Polyhydroxyalkanoate-graft-Poly(N-isopropylacrylamide).

A thermoresponsive graft copolymer polyhydroxyalkanoate-g-poly(N-isopropylacrylamide) or short as PHA-g-PNIPAm, was successfully synthesized via a three-step reaction. First, PNIPAm oligomer with a trithiocarbonate-based chain transfer agent (CTA), short as PNIPAm-CTA, with designed polymerization degree was synthesized via reversible addition-fragmentation chain transfer (RAFT) polymerization. Subsequently, the PNIPAm-CTA was treated with n-butylamine for aminolysis in order to obtain a pendant thiol group at the end of the chain (PNIPAm-SH). Finally, the PNIPAm-SH was grafted onto unsaturated P(3HDD-co-3H10U), a random copolymer of 3-hydroxydodecanoate (3HDD) and 3-hydroxy-10-undecylenate (3H10U), via a thiol-ene click reaction. Enhanced hydrophilicity and thermoresponsive property of the resulted PHA-g-PNIPAm were confirmed by water contact angle studies. The biocompatibility of PHA-g-PNIPAm was comparable to poly-3-hydroxybutyrate (PHB). The graft copolymer PHA-g-PNIPAm based on biopolyester PHA could be a promising material for biomedical applications.

Environmental biodegradation of haloarchaea-produced poly(3-hydroxybutyrate-co-3-hydroxyvalerate) in activated sludge.

Novel poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBHV) copolymers produced by haloarchaea are excellent candidate biomaterials. However, there is no report hitherto focusing on the biodegradation of PHBHV synthesized by haloarchaea. In this study, an environmental biodegradation of haloarchaea-produced PHBHV films, with 10~60 mol% 3-hydroxyvalerate (3HV) composition and different microchemical structures, was studied in nutrition-depleted activated sludge. The changes in mass, molar mass, chemical composition, thermal properties, and surface morphology were monitored. The mass and molar mass of each film decreased significantly, while the PHA monomer composition remained unchanged with time. Interestingly, the sample of random copolymer PHBHV-2 (R-PHBHV-2) (3HV, 30 mol%) had the lowest crystallinity and was degraded faster than R-PHBHV-3 containing the highest 3HV content or the higher-order copolymer PHBHV-1 (O-PHBHV-1) possessing the highest surface roughness. The order of biodegradation rate was in the opposite trend to the degree of crystallizability of the films. Meanwhile, thermal degradation temperature of most films decreased after biodegradation. Additionally, the surface erosion of films was confirmed by scanning electron microscopy. The dominant bacteria probably responsible for the degradation process were identified in the activated sludge. It was inferred that the degradation rate of haloarchaea-produced PHBHV films mainly depended on sample crystallinity, which was determined by monomer composition and microchemical structure and in turn strongly influenced surface morphology.

Production of poly(3-hydroxypropionate) and poly(3-hydroxybutyrate-co-3-hydroxypropionate) from glucose by engineering Escherichia coli.

Poly(3-hydroxypropionate) (P3HP) is the strongest family member of microbial polyhydroxyalkanoates (PHA) synthesized by bacteria grown on 1,3-propandiol or glycerol. In this study synthesis pathways of P3HP and its copolymer P3HB3HP of 3-hydroxybutyrate (3HB) and 3-hydroxypropionate (3HP) were assembled respectively to allow their synthesis from glucose, a more abundant carbon source. Recombinant Escherichia coli was constructed harboring the P3HP synthetic pathway consisting of heterologous genes encoding glycerol-3-phosphate dehydrogenase (gpd1), glycerol-3-P phosphatase (gpp2) from Saccharomyces cerevisiae that catalyzes formation of glycerol from glucose, and genes coding glycerol dehydratase (dhaB123) with its reactivating factors (gdrAB) from Klebsiella pneumoniae that transfer glycerol to 3-hydroxypropionaldehyde, as well as gene encoding propionaldehyde dehydrogenase (pdup) from Salmonella typhimurium which converts 3-hydroxypropionaldehyde to 3-hydroxypropionyl-CoA, together with the gene of PHA synthase (phaC) from Ralstonia eutropha which polymerizes 3-hydroxypropionyl-CoA into P3HP. When phaA and phaB from Ralstonia eutropha respectively encoding ß-ketothiolase and acetoacetate reductase, were introduced into the above P3HP producing recombinant E. coli, copolymers poly(3-hydroxybutyrate-co-3-hydroxypropionate) (P3HB3HP) were synthesized from glucose as a sole carbon source. The above E. coli recombinants grown on glucose LB medium successfully produced 5g/L cell dry weight containing 18% P3HP and 42% P(3HB-co-84mol% 3HP), respectively, in 48h shake flask studies.

Dendrimers in Medicine: Therapeutic Concepts and Pharmaceutical Challenges.

Dendrimers are three-dimensional macromolecular structures originating from a central core molecule and surrounded by successive addition of branching layers (generation). These structures exhibit a high degree of molecular uniformity, narrow molecular weight distribution, tunable size and shape characteristics, as well as multivalency. Collectively, these physicochemical characteristics together with advancements in design of biodegradable backbones have conferred many applications to dendrimers in formulation science and nanopharmaceutical developments. These have included the use of dendrimers as pro-drugs and vehicles for solubilization, encapsulation, complexation, delivery, and site-specific targeting of small-molecule drugs, biopharmaceuticals, and contrast agents. We briefly review these advances, paying particular attention to attributes that make dendrimers versatile for drug formulation as well as challenging issues surrounding the future development of dendrimer-based medicines.

Modulatory effect of human plasma on the internal nanostructure and size characteristics of liquid-crystalline nanocarriers.

The inverted-type liquid-crystalline dispersions comprising cubosomes and hexosomes hold much potential for drug solubilization and site-specific targeting on intravenous administration. Limited information, however, is available on the influence of plasma components on nanostructural and morphological features of cubosome and hexosome dispersions, which may modulate their stability in the blood and their overall biological performance. Through an integrated approach involving SAXS, cryo-TEM, and nanoparticle tracking analysis (NTA) we have studied the time-dependent effect of human plasma (and the plasma complement system) on the integrity of the internal nanostructure, morphology, and fluctuation in size distribution of phytantriol (PHYT)-based nonlamellar crystalline dispersions. The results indicate that in the presence of plasma the internal nanostructure undergoes a transition from the biphasic phase (a bicontinuous cubic phase with symmetry Pn3m coexisting with an inverted-type hexagonal (H2) phase) to a neat hexagonal (H2) phase, which decreases the median particle size. These observations were independent of a direct effect by serum albumin and dispersion-mediated complement activation. The implication of these observations in relation to soft nanocarrier design for intravenous drug delivery is discussed.

Differential Modulation of Cellular Bioenergetics by Poly(L-lysine)s of Different Molecular Weights.

Poly(L-lysine)s (PLLs), and related derivatives, have received considerable attention as nonviral vectors. High molecular weight PLLs (H-PLLs) are superior transfectants compared with low Mw PLLs (L-PLLs), but suggested to be more cytotoxic. Through a pan-integrated metabolomic approach using Seahorse XF technology, we studied the impact of PLL size on cellular bioenergetic processes in two human cell lines. In contrast to L-PLLs (1-5 kDa), H-PLLs (15-30 kDa) were more detrimental to both mitochondrial oxidative phosphorylation (OXPHOS) and glycolytic activity resulting in considerable intracellular ATP depletion, thereby initiating necrotic-type cell death. The cellular differences to polycation sensitivity were further related to the mitochondrial state, where the impact was substantial on cells with hyperpolarized mitochondria. These medium-throughput approaches offer better opportunities for understanding inter-related intracellular and cell type-dependent processes instigating a bioenergetics crisis, thus, aiding selection (from available libraries) and improved design of safer biodegradable polycations for nucleic acid compaction and cell type-specific delivery.

Biosynthesis, characterization, and hemostasis potential of tailor-made poly(3-hydroxybutyrate-co-3-hydroxyvalerate) produced by Haloferax mediterranei.

We report the biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) random copolymers (R-PHBV) or higher-order copolymers (O-PHBV) in Haloferax mediterranei, with adjustable 3-hydroxyvalerate (3HV) incorporation by cofeeding valerate with glucose. Their microchemical structure, molecular weight and its distribution, and thermal and mechanical properties were characterized by NMR, GPC, DSC, TGA, and universal testing machine, respectively. (13)C NMR studies showed that O-PHBV copolymers consisted of short segments of PHB and PHV covalently linked together with random PHBV segments. Consistently, two Tg were observed in the DSC curves of O-PHBV. The "blocky" feature of O-PHBV enhanced crystallinity percentages and improved Young's modulus. Notably, the film of one O-PHBV copolymer, O-PHBV-1, showed unique foveolar cluster-like surface morphology with high hydrophobicity and roughness, as characterized using static contact angle and SEM and AFM analyses. It also exhibited increased platelet adhesion and accelerated blood clotting. The excellent hemostatic properties endow this copolymer with great potential in wound healing.

Live-cell fluorescent microscopy platforms for real-time monitoring of polyplex-cell interaction: basic guidelines.

A myriad of cationic polymeric delivery vehicles are currently being developed with the aim of transporting various forms of nucleic acids to mammalian cells. The complexes between polycations and nucleic acids are referred to as polyplexes. The screening for successful polyplex candidates requires interdisciplinary research platforms and techniques for a more profound understanding of biophysical properties of delivery vehicles and their biological performance, including stability, transfection efficacy and possible cytotoxicity. Fluorescent microscopy has proven to be a useful tool for real-time monitoring of performance and intracellular trafficking of polyplexes as well as for assessing cell functionality. This review highlights the application of some of the most promising fluorescent microscopy platforms in relation to polyplex-mediated transfection processes.

High resolution respirometry analysis of polyethylenimine-mediated mitochondrial energy crisis and cellular stress: Mitochondrial proton leak and inhibition of the electron transport system.

Polyethylenimines (PEIs) are highly efficient non-viral transfectants, but can induce cell death through poorly understood necrotic and apoptotic processes as well as autophagy. Through high resolution respirometry studies in H1299 cells we demonstrate that the 25kDa branched polyethylenimine (25k-PEI-B), in a concentration and time-dependent manner, facilitates mitochondrial proton leak and inhibits the electron transport system. These events were associated with gradual reduction of the mitochondrial membrane potential and mitochondrial ATP synthesis. The intracellular ATP levels further declined as a consequence of PEI-mediated plasma membrane damage and subsequent ATP leakage to the extracellular medium. Studies with freshly isolated mouse liver mitochondria corroborated with bioenergetic findings and demonstrated parallel polycation concentration- and time-dependent changes in state 2 and state 4o oxygen flux as well as lowered ADP phosphorylation (state 3) and mitochondrial ATP synthesis. Polycation-mediated reduction of electron transport system activity was further demonstrated in 'broken mitochondria' (freeze-thawed mitochondrial preparations). Moreover, by using both high-resolution respirometry and spectrophotometry analysis of cytochrome c oxidase activity we were able to identify complex IV (cytochrome c oxidase) as a likely specific site of PEI mediated inhibition within the electron transport system. Unraveling the mechanisms of PEI-mediated mitochondrial energy crisis is central for combinatorial design of safer polymeric non-viral gene delivery systems.

Microbial synthesis of functional homo-, random, and block polyhydroxyalkanoates by ß-oxidation deleted Pseudomonas entomophila.

Functional polyhydroxyalkanoates (PHAs) allow chemical modifications to widen PHA diversity, promising to increase values of these biodegradable and biocompatible polyesters. Among functional PHAs, unsaturated PHA site chains can be easily grafted to add chemical groups, and to cross-link with other PHA polymer chains. However, it has been very difficult to obtain structurally controllable functional homo-, random, or block PHA. For the first time, a ß-oxidation deleted Pseudomonas entomophila was used to successfully synthesize random copolymers of 3-hydroxydodecanoate (3HDD) and 3-hydroxy-9-decenoate (3H9D). Compositions of the random copolymers P(3HDD-co-3H9D) can be adjusted by ratios of dodecanoic acid (DDA) to 9-decenol (9DEO) fed to the culture of P. entomophila. Homopolymer P3H9D was formed when only 9DEO was added to the culture. Diblock copolymers of P3HDD-b-P3H9D were produced by feeding DDA as the first precursor to form a P3HDD block followed by adding 9DEO as the second precursor to form a second P3H9D block. It was demonstrated that random copolymers P(3HDD-co-3H9D) could be crossed-linked under UV-radiation due to the presence of the unsaturated bonds. Thermal and mechanical characterizations of the above homo-, random, and diblock PHA polymers were conducted. It was found that the diblock polymer P3HDD-b-P3H9D increased at least 2-fold on Young's modulus compared with its random copolymers consisting of similar 3HDD/3H9D ratios. This study demonstrates that PHA functionality could be controlled to meet various requirements.

Mechanisms of complement activation by dextran-coated superparamagnetic iron oxide (SPIO) nanoworms in mouse versus human serum.

BACKGROUND: The complement system is a key component of innate immunity implicated in the neutralization and clearance of invading pathogens. Dextran coated superparamagnetic iron oxide (SPIO) nanoparticle is a promising magnetic resonance imaging (MRI) contrast agent. However, dextran SPIO has been associated with significant number of complement-related side effects in patients and some agents have been discontinued from clinical use (e.g., Feridex™). In order to improve the safety of these materials, the mechanisms of complement activation by dextran-coated SPIO and the differences between mice and humans need to be fully understood. METHODS: 20 kDa dextran coated SPIO nanoworms (SPIO NW) were synthesized using Molday precipitation procedure. In vitro measurements of C3 deposition on SPIO NW using sera genetically deficient for various components of the classical pathway (CP), lectin pathway (LP) or alternative pathway (AP) components were used to study mechanisms of mouse complement activation. In vitro measurements of fluid phase markers of complement activation C4d and Bb and the terminal pathway marker SC5b-C9 in normal and genetically deficient sera were used to study the mechanisms of human complement activation. Mouse data were analyzed by non-paired t-test, human data were analyzed by ANOVA followed by multiple comparisons with Student-Newman-Keuls test. RESULTS: In mouse sera, SPIO NW triggered the complement activation via the LP, whereas the AP contributes via the amplification loop. No involvement of the CP was observed. In human sera the LP together with the direct enhancement of the AP turnover was responsible for the complement activation. In two samples out of six healthy donors there was also a binding of anti-dextran antibodies and C1q, suggesting activation via the CP, but that did not affect the total level of C3 deposition on the particles. CONCLUSIONS: There were important differences and similarities in the complement activation by SPIO NW in mouse versus human sera. Understanding the mechanisms of immune recognition of nanoparticles in mouse and human systems has important preclinical and clinical implications and could help design more efficient and safe nano-formulations.

Hyaluronic acid methacryloyl/chitosan methacryloyl/3-methacrylamidophenylboronic acid multifunctional hydrogel loading exosome for diabetic wound healing.

The management of diabetic wounds presents a formidable challenge in clinical settings due to elevated glucose levels, drug-resistant bacterial infections, and a deficiency of bioactive molecules in the microenvironment. To address this challenge, a glucose-responsive Hyaluronic acid Methacryloyl (HAMA)-Chitosan Methacryloyl (CSMA)-3-Methacrylamidophenylboronic acid (MPBA) loading Exosome (H-C-M@Exo) multifunctional hydrogel has been developed to enhance diabetic wound healing in this study. The H-C-M@Exo hydrogel exhibits remarkable features, including high hydrophilicity, excellent pressure resistance, antibacterial properties, and good biocompatibility. It uniquely possesses the ability to sustain the release of exosomes in response to glucose fluctuations. The H-C-M@Exo hydrogel can effectively reduce inflammation, significantly promote rapid re-epithelization, facilitate favorable collagen deposition, and stimulate abundant angiogenesis at the wound sites. Furthermore, the hydrogel induces localized regulation of M2 macrophage polarization, synergistically promoting diabetic angiogenesis and wound healing. These findings suggest H-C-M@Exo hydrogel is a promising biomaterial for the treatment of chronic diabetic wounds.