Optimization of Lipid Nanoparticles for Intramuscular Administration of mRNA Vaccines.

mRNA vaccines have the potential to tackle many unmet medical needs that are unable to be addressed with conventional vaccine technologies. A potent and well-tolerated delivery technology is integral to fully realizing the potential of mRNA vaccines. Pre-clinical and clinical studies have demonstrated that mRNA delivered intramuscularly (IM) with first-generation lipid nanoparticles (LNPs) generates robust immune responses. Despite progress made over the past several years, there remains significant opportunity for improvement, as the most advanced LNPs were designed for intravenous (IV) delivery of siRNA to the liver. Here, we screened a panel of proprietary biodegradable ionizable lipids for both expression and immunogenicity in a rodent model when administered IM. A subset of compounds was selected and further evaluated for tolerability, immunogenicity, and expression in rodents and non-human primates (NHPs). A lead formulation was identified that yielded a robust immune response with improved tolerability. More importantly for vaccines, increased innate immune stimulation driven by LNPs does not equate to increased immunogenicity, illustrating that mRNA vaccine tolerability can be improved without affecting potency.

Fructose-Enhanced Antimicrobial Activity of Silver Nanoparticle-Embedded Polymersome Nanocarriers.

In recent years, an increasing body of research has indicated that the antimicrobial activity of certain antibiotic drugs can be enhanced by the addition of specific metabolites. This study aimed to incorporate these findings into polymersomes (novel polymer-based nanoscale drug delivery vehicles) which can be loaded with various therapeutic molecules and nanoparticles. Polymersome technology has shown promising results in treating antibiotic-resistant infections by co-encapsulating the antibiotic methicillin with silver nanoparticles. Here, silver nanoparticle-embedded polymersomes (AgPs) were synthesized in a similar fashion with gentamicin replacing methicillin as the antibiotic agent and supplemented with fructose to promote efficacy. Two clinically-isolated strains of methicillin-resistant Staphylococcus aureus (MRSA) (ATCC #43300 and ATCC #25923) were cultured and treated with the new AgP formulations, with the former strain being susceptible to gentamicin and the latter strain being resistant to gentamicin. The treatment of the non-resistant strain yielded promising results with the polymersomes without fructose supplementation inducing a maximal growth rate reduction of up to 40% and an increase in lag time of up to 141% relative to the untreated control. Impressively, the fructose-loaded polymersomes completely eliminated the bacterial growth over the observed time period at the higher doses and outperformed the no-fructose treatment at all concentrations. However, despite significantly reducing bacterial growth, the treatment of the gentamicin-resistant strain did not seem to be enhanced by the addition of fructose. Lastly, the present study demonstrated that the presence of fructose in the polymersomes seemed to slightly ameliorate the cytotoxic effect of the treatment on human dermal fibroblasts (a model mammalian cell). In addition to developing and testing a new polymersome formulation with fructose resulting in increased efficacy, the results of this study also demonstrated the variability inherent to developing novel antimicrobial treatments for different bacterial strains.

Engineering Adhesive and Antimicrobial Hyaluronic Acid/Elastin-like Polypeptide Hybrid Hydrogels for Tissue Engineering Applications.

Hydrogel-based biomaterials have been widely used for tissue engineering applications because of their high water content, swellability, and permeability, which facilitate transport and diffusion of essential nutrients, oxygen, and waste across the scaffold. These characteristics make hydrogels suitable for encapsulating cells and creating a cell supportive environment that promotes tissue regeneration when implanted in vivo. This is particularly important in the context of tissues whose intrinsic regenerative capacity is limited, such as cartilage. However, the clinical translation of hydrogels has been limited by their poor mechanical performance, low adhesive strength, uncontrolled degradation rates, and their susceptibility to bacterial colonization. Here, we introduce an elastic, antimicrobial, and adhesive hydrogel comprised of methacrylated hyaluronic acid (MeHA) and an elastin-like polypeptide (ELP), which can be rapidly photo-cross-linked in situ for the regeneration and repair of different tissues. Hybrid hydrogels with a wide range of physical properties were engineered by varying the concentrations of MeHA and ELP. In addition, standard adhesion tests demonstrated that the MeHA/ELP hydrogels exhibited higher adhesive strength to the tissue than commercially available tissue adhesives. MeHA/ELP hydrogels were then rendered antimicrobial through the incorporation of zinc oxide (ZnO) nanoparticles, and were shown to significantly inhibit the growth of methicillin-resistant Staphylococcus aureus (MRSA), as compared to controls. Furthermore, the composite adhesive hydrogels supported in vitro mammalian cellular growth, spreading, and proliferation. In addition, in vivo subcutaneous implantation demonstrated that MeHA/ELP hydrogels did not elicit any significant inflammatory response, and could be efficiently biodegraded while promoting the integration of new autologous tissue. In summary, we demonstrated for the first time that MeHA/ELP-ZnO hydrogel can be used as an adhesive and antimicrobial biomaterial for tissue engineering applications, because of its highly tunable physical characteristics, as well as remarkable adhesive and antimicrobial properties.

Killing malignant melanoma cells with protoporphyrin IX-loaded polymersome-mediated photodynamic therapy and cold atmospheric plasma.

Traditional cancer treatments contain several limitations such as incomplete ablation and multidrug resistance. It is known that photodynamic therapy (PDT) is an effective treatment for several tumor types especially melanoma cells. During the PDT process, protoporphyrin IX (PpIX), an effective photosensitizer, can selectively kill cancer cells by activating a special light source. When tumor cells encapsulate a photosensitizer, they can be easily excited into an excited state by a light source. In this study, cold atmospheric plasma (CAP) was used as a novel light source. Results of some studies have showed that cancer cells can be effectively killed by using either a light source or an individual treatment due to the generation of reactive oxygen species and electrons from a wide range of wavelengths, which suggest that CAP can act as a potential light source for anticancer applications compared with UV light sources. Results of the present in vitro study indicated for the first time that PpIX can be successfully loaded into polymersomes. Most importantly, cell viability studies revealed that PpIX-loaded polymersomes had a low toxicity to healthy fibroblasts (20% were killed) at a concentration of 400 µg/mL, but they showed a great potential to selectively kill melanoma cells (almost 50% were killed). With the application of CAP posttreatment, melanoma cell viability significantly decreased (80% were killed) compared to not using a light source (45% were killed) or using a UV light source (65% were killed). In summary, these results indicated for the first time that PpIX-loaded polymersomes together with CAP posttreatment could be a promising tool for skin cancer drug delivery with selective toxicity toward melanoma cells sparing healthy fibroblasts.

pH-Controlled Cerium Oxide Nanoparticle Inhibition of Both Gram-Positive and Gram-Negative Bacteria Growth.

Here, the antibacterial activity of dextran-coated nanoceria was examined against Pseudomonas aeruginosa and Staphylococcus epidermidis by varying the dose, the time of treatment, and the pH of the solution. Findings suggested that dextran-coated nanoceria particles were much more effective at killing P. aeruginosa and S. epidermidis at basic pH values (pH = 9) compared to acidic pH values (pH = 6) due to a smaller size and positive surface charge at pH 9. At pH 9, different particle concentrations did cause a delay in the growth of P. aeruginosa, whereas impressively S. epidermidis did not grow at all when treated with a 500 µg/mL nanoceria concentration for 24 hours. For both bacteria, a 2 log reduction and elevated amounts of reactive oxygen species (ROS) generation per colony were observed after 6 hours of treatment with nanoceria at pH 9 compared to untreated controls. After 6 hours of incubation with nanoceria at pH 9, P. aeruginosa showed drastic morphological changes as a result of cellular stress. In summary, this study provides significant evidence for the use of nanoceria (+4) for a wide range of anti-infection applications without resorting to the use of antibiotics, for which bacteria are developing a resistance towards anyway.

High throughput microencapsulation of Bacillus subtilis in semi-permeable biodegradable polymersomes for selenium remediation.

Encapsulating bacteria within constrained microenvironments can promote the manifestation of specialized behaviors. Using double-emulsion droplet-generating microfluidic synthesis, live Bacillus subtilis bacteria were encapsulated in a semi-permeable membrane composed of poly(ethylene glycol)-b-poly(D,L-lactic acid) (mPEG-PDLLA). This polymer membrane was sufficiently permeable to permit exponential bacterial growth, metabolite-induced gene expression, and rapid biofilm growth. The biodegradable microparticles retained structural integrity for several days and could be successfully degraded with time or sustained bacterial activity. Microencapsulated B. subtilis successfully captured and contained sodium selenite added outside the polymersomes, converting the selenite into elemental selenium nanoparticles that were selectively retained inside the polymer membrane. This remediation of selenium using polymersomes has high potential for reducing the toxicity of environmental selenium contamination, as well as allowing selenium to be harvested from areas not amenable to conventional waste or water treatment.

Superparamagnetic iron oxide-encapsulating polymersome nanocarriers for biofilm eradication.

The rising prevalence and severity of antibiotic-resistant biofilm infections poses an alarming threat to public health worldwide. Here, biocompatible multi-compartment nanocarriers were synthesized to contain both hydrophobic superparamagnetic iron oxide nanoparticles (SPIONs) and the hydrophilic antibiotic methicillin for the treatment of medical device-associated infections. SPION co-encapsulation was found to confer unique properties, enhancing both nanocarrier relaxivity and magneticity compared to individual SPIONs. These iron oxide-encapsulating polymersomes (IOPs) penetrated 20 µm thick Staphylococcus epidermidis biofilms with high efficiency following the application of an external magnetic field. Three-dimensional laser scanning confocal microscopy revealed differential bacteria death as a function of drug and SPION loading. Complete eradication of all bacteria throughout the biofilm thickness was achieved using an optimized IOP formulation containing 40 µg/mL SPION and 20 µg/mL of methicillin. Importantly, this formulation was selectively toxic towards methicillin-resistant biofilm cells but not towards mammalian cells. These novel iron oxide-encapsulating polymersomes demonstrate that it is possible to overcome antibiotic-resistant biofilms by controlling the positioning of nanocarriers containing two or more therapeutics.

Reducing Staphylococcus aureus growth on Ti alloy nanostructured surfaces through the addition of Sn.

ß-type Ti alloys containing Nb are exciting materials for numerous orthopedic and dental applications due to their exceptional mechanical properties. To improve their cytocompatibility properties (such as increasing bone growth and decreasing infection), the surfaces of such materials can be optimized by adding elements and/or nanotexturing through anodization. Because of the increasing prevalence of orthopedic implant infections, the objective of this in vitro study was to add Sn and create unique nanoscale surface features on ß-type Ti alloys. Nanotubes and nanofeatures on Ti-35Nb and Ti-35Nb-4Sn alloys were created by anodization in a HF-based electrolyte and then heat treated in a furnace to promote amorphous structures and phases such as anatase, a mixture of anatase-rutile, and rutile. Samples were characterized by SEM, which indicated different morphologies dependent on the oxide content and method of modification. XPS experiments identified the oxide content which resulted in a phase transformation in the oxide layer formed onto Ti-35Nb and Ti-35Nb-4Sn alloys. Most importantly, regardless of the resulting nanostructures (nanotubes or nanofeatures) and crystalline phase, this study showed for the first time that adding Sn to ß-type Ti alloys strongly decreased the adhesion of Staphylococcus aureus (S. aureus; a bacteria which commonly infects orthopedic implants leading to their failure). Thus, this study demonstrated that ß-type Ti alloys with Nb and Sn have great promise to improve numerous orthopedic applications where infection may be a concern.

The ability of streptomycin-loaded chitosan-coated magnetic nanocomposites to possess antimicrobial and antituberculosis activities.

Magnetic nanoparticles (MNPs) were synthesized by the coprecipitation of Fe(2+) and Fe(3+) iron salts in alkali media. MNPs were coated by chitosan (CS) to produce CS-MNPs. Streptomycin (Strep) was loaded onto the surface of CS-MNPs to form a Strep-CS-MNP nanocomposite. MNPs, CS-MNPs, and the nanocomposites were subsequently characterized using X-ray diffraction and were evaluated for their antibacterial activity. The antimicrobial activity of the as-synthesized nanoparticles was evaluated using different Gram-positive and Gram-negative bacteria, as well as Mycobacterium tuberculosis. For the first time, it was found that the nanoparticles showed antimicrobial activities against the tested microorganisms (albeit with a more pronounced effect against Gram-negative than Gram-positive bacteria), and thus, should be further studied as a novel nano-antibiotic for numerous antimicrobial and antituberculosis applications. Moreover, since these nanoparticle bacteria fighters are magnetic, one can easily envision magnetic field direction of these nanoparticles to fight unwanted microorganism presence on demand. Due to the ability of magnetic nanoparticles to increase the sensitivity of imaging modalities (such as magnetic resonance imaging), these novel nanoparticles can also be used to diagnose the presence of such microorganisms. In summary, although requiring further investigation, this study introduces for the first time a new type of magnetic nanoparticle with microorganism theranostic properties as a potential tool to both diagnose and treat diverse microbial and tuberculosis infections.

Silver nanoparticle-embedded polymersome nanocarriers for the treatment of antibiotic-resistant infections.

The rapidly diminishing number of effective antibiotics that can be used to treat infectious diseases and associated complications in a physician's arsenal is having a drastic impact on human health today. This study explored the development and optimization of a polymersome nanocarrier formed from a biodegradable diblock copolymer to overcome bacterial antibiotic resistance. Here, polymersomes were synthesized containing silver nanoparticles embedded in the hydrophobic compartment, and ampicillin in the hydrophilic compartment. Results showed for the first time that these silver nanoparticle-embedded polymersomes (AgPs) inhibited the growth of Escherichia coli transformed with a gene for ampicillin resistance (bla) in a dose-dependent fashion. Free ampicillin, AgPs without ampicillin, and ampicillin polymersomes without silver nanoparticles had no effect on bacterial growth. The relationship between the silver nanoparticles and ampicillin was determined to be synergistic and produced complete growth inhibition at a silver-to-ampicillin ratio of 1 : 0.64. In this manner, this study introduces a novel nanomaterial that can effectively treat problematic, antibiotic-resistant infections in an improved capacity which should be further examined for a wide range of medical applications.

Similar healthy osteoclast and osteoblast activity on nanocrystalline hydroxyapatite and nanoparticles of tri-calcium phosphate compared to natural bone.

While there have been numerous studies to determine osteoblast (bone forming cell) functions on nanocrystalline compared to micron crystalline ceramics, there have been few studies which have examined osteoclast activity (including tartrate-resistant acid phosphatase, formation of resorption pits, size of resorption pits, and receptor activator of nuclear factor κB [RANK]). This is despite the fact that osteoclasts are an important part of maintaining healthy bone since they resorb bone during the bone remodeling process. Moreover, while it is now well documented that bone formation is enhanced on nanoceramics compared to micron ceramics, some have pondered whether osteoblast functions (such as osteoprotegerin and RANK ligand [RANKL]) are normal (ie, non-diseased) on such materials compared to natural bone. For these reasons, the objective of the present in vitro study was to determine various functions of osteoclasts and osteoblasts on nanocrystalline and micron crystalline hydroxyapatite as well as tri-calcium phosphate materials and compare such results to cortical and cancellous bone. Results showed for the first time similar osteoclast activity (including tartrate-resistant acid phosphatase, formation of resorption pits, size of resorption pits, and RANK) and osteoblast activity (osteoprotegerin and RANKL) on nanocrystalline hydroxyapatite compared to natural bone, whereas osteoclast and osteoblast functions on micron crystalline versions of these ceramics were much different than natural bone. In this manner, this study provides additional evidence that nanocrystalline calcium phosphates can serve as suitable synthetic analogs to natural bone to improve numerous orthopedic applications. It also provides the first data of healthy osteoclast and osteoblast functions on nanocrystalline calcium phosphates compared to natural bone.

Development of a biocompatible nanodelivery system for tuberculosis drugs based on isoniazid-Mg/Al layered double hydroxide.

The primary challenge in finding a treatment for tuberculosis (TB) is patient non-compliance to treatment due to long treatment duration, high dosing frequency, and adverse effects of anti-TB drugs. This study reports on the development of a nanodelivery system that intercalates the anti-TB drug isoniazid into Mg/Al layered double hydroxides (LDHs). Isoniazid was found to be released in a sustained manner from the novel nanodelivery system in humans in simulated phosphate buffer solutions at pH 4.8 and pH 7.4. The nanodelivery formulation was highly biocompatible compared to free isoniazid against human normal lung and 3T3 mouse fibroblast cells. The formulation was active against Mycobacterium tuberculosis and gram-positive bacteria and gram-negative bacteria. Thus results show significant promise for the further study of these nanocomposites for the treatment of TB.

Antimycobacterial, antimicrobial, and biocompatibility properties of para-aminosalicylic acid with zinc layered hydroxide and Zn/Al layered double hydroxide nanocomposites.

The treatment of tuberculosis by chemotherapy is complicated due to multiple drug prescriptions, long treatment duration, and adverse side effects. We report here for the first time an in vitro therapeutic effect of nanocomposites based on para-aminosalicylic acid with zinc layered hydroxide (PAS-ZLH) and zinc-aluminum layered double hydroxides (PAS-Zn/Al LDH), against mycobacteria, Gram-positive bacteria, and Gram-negative bacteria. The nanocomposites demonstrated good antimycobacterial activity and were found to be effective in killing Gram-positive and Gram-negative bacteria. A biocompatibility study revealed good biocompatibility of the PAS-ZLH nanocomposites against normal human MRC-5 lung cells. The para-aminosalicylic acid loading was quantified with high-performance liquid chromatography analysis. In summary, the present preliminary in vitro studies are highly encouraging for further in vivo studies of PAS-ZLH and PAS-Zn/Al LDH nanocomposites to treat tuberculosis.

Synthesis, characterization, and antimicrobial activity of an ampicillin-conjugated magnetic nanoantibiotic for medical applications.

Because of their magnetic properties, magnetic nanoparticles (MNPs) have numerous diverse biomedical applications. In addition, because of their ability to penetrate bacteria and biofilms, nanoantimicrobial agents have become increasingly popular for the control of infectious diseases. Here, MNPs were prepared through an iron salt coprecipitation method in an alkaline medium, followed by a chitosan coating step (CS-coated MNPs); finally, the MNPs were loaded with ampicillin (amp) to form an amp-CS-MNP nanocomposite. Both the MNPs and amp-CS-MNPs were subsequently characterized and evaluated for their antibacterial activity. X-ray diffraction results showed that the MNPs and nanocomposites were composed of pure magnetite. Fourier transform infrared spectra and thermogravimetric data for the MNPs, CS-coated MNPs, and amp-CS-MNP nanocomposite were compared, which confirmed the CS coating on the MNPs and the amp-loaded nanocomposite. Magnetization curves showed that both the MNPs and the amp-CS-MNP nanocomposites were superparamagnetic, with saturation magnetizations at 80.1 and 26.6 emu g(-1), respectively. Amp was loaded at 8.3%. Drug release was also studied, and the total release equilibrium for amp from the amp-CS-MNPs was 100% over 400 minutes. In addition, the antimicrobial activity of the amp-CS-MNP nanocomposite was determined using agar diffusion and growth inhibition assays against Gram-positive bacteria and Gram-negative bacteria, as well as Candida albicans. The minimum inhibitory concentration of the amp-CS-MNP nanocomposite was determined against bacteria including Mycobacterium tuberculosis. The synthesized nanocomposites exhibited antibacterial and antifungal properties, as well as antimycobacterial effects. Thus, this study introduces a novel ß-lactam antibacterial-based nanocomposite that can decrease fungus activity on demand for numerous medical applications.

Greater fibroblast proliferation on an ultrasonicated ZnO/PVC nanocomposite material.

There has been a significant and growing concern over nosocomial medical device infections. Previous studies have demonstrated that embedding nanoparticles alone (specifically, zinc oxide [ZnO]) in conventional polymers (eg, polyvinyl chloride [PVC]) can decrease bacteria growth and may have the potential to prevent or disrupt bacterial processes that lead to infection. However, little to no studies have been conducted to determine mammalian cell functions on such a nanocomposite material. Clearly, for certain medical device applications, maintaining healthy mammalian cell functions while decreasing bacteria growth is imperative (yet uncommon). For this reason, in the presented study, ZnO nanoparticles of varying sizes (from 10 nm to >200 nm in diameter) and functionalization (including no functionalization to doping with aluminum oxide and functionalizing with a silane coupling agent KH550) were incorporated into PVC either with or without ultrasonication. Results of this study provided the first evidence of greater fibroblast density after 18 hours of culture on the smallest ZnO nanoparticle incorporated PVC samples with dispersion aided by ultrasonication. Specifically, the greatest amount of fibroblast proliferation was measured on ZnO nanoparticles functionalized with a silane coupling agent KH550; this sample exhibited the greatest dispersion of ZnO nanoparticles. Water droplet tests showed a general trend of decreased hydrophilicity when adding any of the ZnO nanoparticles to PVC, but an increase in hydrophilicity (albeit still below controls or pure PVC) when using ultrasonication to increase ZnO nanoparticle dispersion. Future studies will have to correlate this change in wettability to initial protein adsorption events that may explain fibroblast behavior. Mechanical tests also provided evidence of the ability to tailor mechanical properties of the ZnO/PVC nanocomposites through the use of the different ZnO nanoparticles. Coupled with previous antibacterial studies, the present study demonstrated that highly dispersed ZnO/PVC nanocomposite materials should be further studied for numerous medical device applications.

Reduced adhesion of Staphylococcus aureus to ZnO/PVC nanocomposites.

In hospitals and clinics worldwide, medical device surfaces have become a rapidly growing source of nosocomial infections. In particular, patients requiring mechanical ventilation (and, thus, intubation with an endotracheal tube) for extended lengths of time are faced with a high probability of contracting ventilator-associated pneumonia. Once inserted into the body, the endotracheal tube provides a surface to which bacteria can adhere and form a biofilm (a robust, sticky matrix that provides protection against the host immune system and antibiotic treatment). Adding to the severity of this problem is the spread of bacterial genetic tolerance to antibiotics, in part demonstrated by the recent and significant increase in the prevalence of methicillin-resistant Staphylococcus aureus. To combat these trends, different techniques in biomaterial design must be explored. Recent research has shown that nanomaterials (materials with at least one dimension less than 100 nm) may have the potential to prevent or disrupt bacterial processes that lead to infections. In this study, polyvinyl chloride (PVC) taken from a conventional endotracheal tube was embedded with varying concentrations of zinc oxide (ZnO) nanoparticles. S. aureus biofilms were then grown on these nanocomposite surfaces during a 24-hour culture. Following this, biofilms were removed from the surfaces and the number of colony forming units present was assessed. Bacterial proliferation on the samples embedded with the highest concentration of ZnO nanoparticles was 87% less when compared to the control, indicating that this technique is effective at reducing biofilm formation on PVC surfaces without the use of antibiotics.