ABSTRACT Nucleoside and nucleotide analogs are an impactful class of antivirals, yet their efficacy is often limited by inefficient intracellular accumulation and enzymatic activation. Many analogs are administered as nucleoside prodrugs, dependent on host or viral kinases for sequential phosphorylation, with the first monophosphate-forming step widely considered rate-limiting. Medicinal chemistry strategies such as utilizing masked-phosphate prodrugs may improve uptake and activation, but require alternative metabolic processing to generate the active triphosphate. Nanoparticle delivery provides an alternative or complementary approach by directly encapsulating phosphorylated nucleotide analogs. Nanoparticles made from materials including lipids, polymers, dendrimers, or polysaccharides can protect phosphorylated drugs from degradation, improve cellular uptake, and enable controlled or tissue-specific release. This review provides a brief context and overview of recent advances in nanoparticle-mediated delivery of phosphorylated antiviral nucleotides, drawing on parallels from oncology, and evaluates the technological advances and limitations influencing their continued development as antiviral carriers.

Measurement of antibiotic accumulation in bacteria is essential for full understanding of the mechanisms of antimicrobial resistance but requires a highly sensitive analytical assay. A suitable ultra performance reverse phase liquid chromatographic tandem mass spectrometry (UPLC-MS/MS) method was developed in the electrospray negative ionisation mode for quantifying tobramycin in Pseudomonas aeruginosa cell extracts. P. aeruginosa cell lysate extracts were prepared and spiked with tobramycin and kanamycin (internal standard). Following a protein precipitation extraction procedure, the sample was applied to a reverse phase C18 column, equilibrated in 10 mM ammonium hydroxide at pH 11. Tobramycin and kanamycin were eluted using an acetonitrile gradient and detected in the electrospray negative ionisation mode. The retention times for kanamycin and tobramycin were 1.9 and 2.5 min, respectively and total run time of 10 min. The assay demonstrated linearity in the range of 0.02 - 1 mu g mL-1 (R = 0.9999), with limits of detection and quantitation at 1.42 ng mL-1 and 10 ng mL-1, respectively. The precision, expressed as the coefficient of variation, ranged from 1.4 % to 6.5 %, and the accuracy, expressed as bias, ranged from 0.4 % to 17.1 % (ICH, 1996). Using a simple protein precipitation method, the recoveries (%) of tobramycin from cell lysate supernatant for quality controls were 99.7-105.2 % and this result shows that the assay is quantitative. This validated analytical protocol will facilitate future studies aimed at determining the cellular uptake kinetics of tobramycin by P. aeruginosa under various controlled conditions and it could be used for other applications.

Chronic infections of Pseudomonas aeruginosa are commonly treated with tobramycin. During infections, the bacteria can exist under conditions of oxygen deprivation that render them less susceptible to this antibiotic. The aims of this research were to investigate the genetic basis of tobramycin resistance under anaerobic conditions, and to investigate the effects of anaerobiosis and mutations on the cellular concentration of tobramycin. Ten mutants with lowered susceptibility to tobramycin than wild-type bacteria were evolved from a laboratory reference strain under anaerobic conditions. Mutations were identified by genome sequencing. Mutations had arisen most frequently in the fusA1 gene that encodes elongation factor EF-G1A and in genes involved in twitching motility. Cellular concentrations of tobramycin were then measured. Mutations in fusA1 or absence of the MexXY efflux pump that is associated with tobramycin resistance did not alter the cellular tobramycin concentration under either anaerobic or aerobic conditions. Anaerobic growth reduced the cellular concentration of tobramycin, relative to aerobically grown bacteria, in some but not all of five tested P. aeruginosa isolates. Overall, our findings indicate that anaerobiosis and mutations that reduce aminoglycoside effectiveness do not lower the cellular concentration of antibiotic but instead reduce susceptibility through other mechanisms.

Conjugation of a therapeutic agent to a polymer for enhanced delivery into target cells followed by its intracellular triggered release has proved to be an effective drug delivery approach. This approach is applied to the delivery of the immune-stimulatory unmethylated cytosine-phosphate-guanine (CpG) oligonucleotide for an anti-tumour immune response after intratumoral administration. On average four CpG-1668 molecules were covalently linked to a 40-kDa amino-functionalised dextran polymer via either a non-reversible (CpG-dextran) or an intracellular redox-responsive disulfide linkage (CpG-SS-dextran). Dynamic light scattering analysis showed that both conjugates had a similar particle size and surface charge of 17 nm and-10 mV, respectively. Agarose gel electrophoresis analysis showed that CpG-SS-dextran was stable in the extracellular low glutathione (GSH) concentration range (i.e. 10-20 mu M) and was cleaved at the higher intracellular GSH concentration (5 mM), while CpG-dextran was stable in both GSH concentrations. Uptake and activation assays on bone-marrow- derived dendritic cells showed no significant difference between free CpG, CpG-dextran and CpG-SS-dextran. In a mouse subcutaneous colorectal tumour model the CpG-SS-dextran showed a statistically significantly greater inhibition of tumour growth (p < 0.03) and prolonged survival (p < 0.001) compared to CpG-dextran or free CpG. These results demonstrate that the redox-triggered intracellular release of CpG from a dextran polymer carrier has promise for intratumoral therapeutic vaccination against cancer.

There is a need to find alternative solvents for electrospinning that are less hazardous and more environmentally safe than the Class II solvents currently used. We characterized poly(lactic-co-glycolic acid) (PLGA) nanofibers (NFs) electrospun using a new, less toxic solvent system comprised the Class III solvents acetone:ethyl acetate (ACE:EtAc). Both solvent systems produced amorphous NFs. Fourier-transform infrared spectroscopy (FTIR) showed no change in PLGA composition after electrospinning. ACE:EtAc NFs showed a 53% burst release of paracetamol, followed by a gradual release not statistically different to release from DCM:DMF. ACE:EtAc can be used as a solvent to electrospin PLGA NFs however, with larger fiber diameter.

The effect of dextran molecular weight on the in vitro physicochemical and immune properties of cytosine-phosphate-guanine (CpG) oligodeoxynucleotide-amino-dextran conjugates is investigated. CpG-1668 was conjugated at the 3'-end to amino-dextran of differing molecular weight (20, 40, 70 or 110-kDa) via a stable bis-aryl hydrazone linkage. Conjugate formation was confirmed by agarose gel electrophoresis and dynamic light scattering measured the size and surface charge of conjugates. Uptake and immune-stimulatory activity of CpG-dextran by antigen-presenting cells was evaluated by flow cytometry and confocal microscopy. Degradation by DNase I was monitored by loss of the fluorescent signal from labelled CpG and changes in size and zeta potential. Hydrazone bond formation (UV 354 nm) showed on average four CpG molecules conjugated per polymer. CpG-dextran prepared from 20 or 40-kDa dextran had a size of 17 nm while 70 or 110-kDa was 30 nm. CpG-dextran was preferentially taken up by dendritic cells, followed by macrophages and then B-cells. Only the 20-kDa dextran conjugate significantly enhanced uptake by bone-marrow derived dendritic cells (BMDCs) compared to free CpG. Confocal microscopy showed that CpG and CpG-dextran accumulates in the endo-lysosomal compartment of BMDCs at 24 h. All conjugates upregulated activation markers (CD40, CD80 or CD86) of BMDCs to a similar level as for free CpG. CpG-dextran 40-kDa produced highest levels of cytokines (TNF-α, IL-6, and IL-12p70) secreted by BMDCs. Enzymatic protection assays showed that the conjugate made from dextran 20-kDa provided no protection for CpG while the higher molecular weight conjugates reduced degradation by DNase I. The 40-kDa dextran conjugate produced the greatest in vitro immune activity, this was due to the conjugate being relatively small in size for cell uptake while sufficiently large enough to protect CpG from nuclease attack. These in vitro studies identify the need to consider the molecular weight of the carrier in bioconjugate design.

Fertiliser has been a vital part of agriculture due to it boosting crop productivity and preventing starvation throughout the world. Despite this huge contribution, the application of nitrogen (N) fertilisers results in N leaching and the formation of greenhouse gases, which threaten the environment and human health. To minimise the impacts, slow/controlled release fertilisers (S/CRFs) have been being developed since the beginning of the 20th century. Despite the efforts made over a century, the basic terminological and classification information of these fertilisers remains vague. The scientific knowledge published in S/CRF patents has also been overlooked since the beginning. This review focused on the information gaps, clarified the definitions, differentiation and classification methods that have been randomly used in previous literature. The objectives, formulations and technologies of 109 controlled release urea patents involving sulphur coated urea, polymer coated urea and urea matrix fertilisers published in the years since these products emerged were also reviewed to 1) highlight the overlooked scientific knowledge in the patents; 2) understand the evolutionary processes and current research states of the products; 3) clarify research preferences and challenges to date; 4) identify remaining gaps for the future direction. It is expected that the organised basic information and the patent knowledge highlighted in this paper can be new resources and foster the development of S/CRFs in the future.

Biologics can be combined with liquid polymer materials and electrospun to produce a dry nanofibrous scaffold. Unlike spray-drying and freeze-drying, electrospinning minimizes the physiological stress on sensitive materials, and nanofiber mat properties such as hydrophobicity, solubility, and melting temperature can be tuned based on the polymer composition. In this study, we explored the dry formulation of a virus-like particle (VLP) vaccine by electrospinning VLP derived from rabbit hemorrhagic disease virus modified to carry the MHC-I gp100 tumor-associated antigen epitope. VLP were added to a polyvinylpyrrolidone (PVP) solution (15% w/v) followed by electrospinning at 24 kV. Formation of a nanofibrous mat was confirmed by scanning electron microscopy, and the presence of VLP was confirmed by transmission electron microscopy and Western blot. VLP from the nanofibers induced T-cell activation and interferon- (IFN-) γ production in vitro. To confirm in vivo cytotoxicity, Pmel mice treated by injection with gp100 VLP from nanofibers induced a gp100 specific immune response, lysing approximately 65% of gp100-pulsed target cells, comparable to mice vaccinated with gp100 VLP in PBS. VLP from nanofibers also induced an antibody response. This work shows that electrospinning can be used to dry-formulate VLP, preserving both humoral and cell-mediated immunity.

Electrospun nanofibers have been extensively studied for encapsulated drugs releasing from the inside of the fiber matrix, but have been barely looked at for their potential to control release as a semi-permeable membrane. This study investigated molecular transport behaviors across nanofiber membranes with different micro-structure sizes and compositions. Four types of membranes were made by 5% and 10% poly (ε-caprolactone) (PCL) solutions electro-spun with or without 50 nm calcium carbonate (CaCO3) nanoparticles. The membranes were tested for thickness, fiber diameter, pore size, porosity, tensile strength and elongation, contact angle of water and their impacts on molecular transport behaviors. The presence of the CaCO3 nanoparticles made the 5% membranes stronger and stiffer but the 10% membranes weaker and less stiff due to the different (covering or embedded) locations of the nanoparticles with the corresponding fibers. Solute transport studies using caffeine as the model drug found the 5% membranes further retarded release from the 10% membranes, regardless of only half the amount of material being used for synthesis. The addition of CaCO3 nanoparticles aided the water permeation process and accelerated initial transports. The difference in release profiles between 5% and 10% membranes suggests different release mechanisms, with membrane-permeability dominated release for 5% PCL membranes and solute-concentration-gradient dominated release for 10% PCL membranes.

Cytosine-phosphate-guanine (CpG) oligonucleotides are commonly-used vaccine adjuvants to promote the activation of antigen-presenting cells (APCs). To mount an effective immune response, CpG needs to be internalized and bind to its endosomal Toll-like receptor 9 (TLR-9) inside the APCs. Using flow cytometry and fluorescence microscopy, this article presents the cellular uptake data of the amino-dextran nanoparticle (aDNP) and aDNP loaded with CpG immobilized on its surface by either electrostatic adsorption or covalent conjugation. The uptake of fluorescently-labelled aDNPs by murine splenic dendritic cells and macrophages was determined by flow cytometry and uptake by murine bone-marrow-derived dendritic cells was evaluated by fluorescence microscopy. The data presented in this paper correlates with the in vitro immune-stimulatory activity observed for the two different CpG loading methods in the research article "Nanoparticle system based on amino-dextran as a drug delivery vehicle: immune-stimulatory CpG-oligonucleotide loading and delivery" (Nguyen et al., 2020) [1]. The data provide experimental evidence for a better understanding how the nanoparticle surface loading method of CpG influences the uptake of these nanoparticles by antigen-presenting cells as a step guide in the design of more effective vaccine formulations.

Breast cancer (BC) is the most frequently diagnosed cancer in women, with many patients experiencing recurrence following treatment. Antigens delivered on virus-like particles (VLPs) induce a targeted immune response and here we investigated whether the co-delivery of multiple antigens could induce a superior anti-cancer response for BC immunotherapy. VLPs were designed to recombinantly express murine survivin and conjugated with an aberrantly glycosylated mucin-1 (MUC1) peptide using an intracellular cleavable bis-arylhydrazone linker. Western blotting, electron microscopy and UV absorption confirmed survivin-VLP expression and MUC1 conjugation. To assess the therapeutic efficacy of VLPs, orthotopic BC tumours were established by injecting C57mg.MUC1 cells into the mammary fat pad of mice, which were then vaccinated with surv.VLP-SS-MUC1 or VLP controls. While wild-type mice vaccinated with surv.VLP-SS-MUC1 showed enhanced survival compared to VLPs delivering either antigen alone, MUC1 transgenic mice vaccinated with surv.VLP-SS-MUC1 showed no enhanced survival compared to controls. Hence, while co-delivery of two tumour antigens on VLPs can induce a superior anti-tumour immune response compared to the delivery of single antigens, additional strategies must be employed to break tolerance when targeted tumour antigens are expressed as endogenous self-proteins. Using VLPs for the delivery of multiple antigens represents a promising approach to improving BC immunotherapy, and has the potential to be an integral part of combination therapy in the future.

Compression-induced destabilization was investigated in various celecoxib amorphous solid dispersions containing hydroxypropyl methylcellulose (HPMC), poly(vinylpyrrolidone)/vinyl acetate copolymer (PVP/VA), or poly(vinylpyrrolidone) (PVP) at a concentration range of 1-10% w/w. Pharmaceutically relevant (125 MPa pressure with a minimal dwell time) and extreme (500 MPa pressure with a 60 s dwell time) compression conditions were applied to these systems, and the changes in their physical stability were monitored retrospectively (i.e., in the supercooled state) using dynamic differential scanning calorimetry (DSC) and low-frequency Raman (LFR) measurements over a broad temperature range (-90 to 200 and -150 to 140 °C, respectively). Both techniques revealed similar changes in the crystallization behavior between samples, where the application of a higher compression force of 500 MPa resulted in a more pronounced destabilization effect that was progressively mitigated with increasing polymer content. However, other aspects such as more favorable intermolecular interactions did not appear to have any effect on reducing this undesirable effect. Additionally, for the first time, LFR spectroscopy was used as a viable technique to determine the secondary or local glass-transition temperature, Tg,β, a major indicator of the physical stability of neat amorphous pharmaceutical systems.

A series of C-2 derivatized 8-sulfonamidoquinolines were evaluated for their antibacterial activity against the common mastitis causative pathogens Streptococcus uberis, Staphylococcus aureus and Escherichia coli, both in the presence and absence of supplementary zinc (50 mu M ZnSO4). The vast majority of compounds tested were demonstrated to be significantly more active against S. uberis when in the presence of supplementary zinc (MICs as low as 0.125 mu g/mL were observed in the presence of 50 mu M ZnSO4). Compounds 5, 34-36, 39, 58, 79, 82, 94 and 95 were shown to display the greatest antibacterial activity against S. aureus (MIC <= 8 mu g/mL; both in the presence and absence of supplementary zinc), while compounds 56, 58 and 66 were demonstrated to also exhibit activity against E. coli (MIC <= 16 mu g/mL; under all conditions). Compounds 56, 58 and 66 were subsequently confirmed to be bactericidal against all three mastitis pathogens studied, with MBCs (>= 3log(10) CFU/mL reduction) of <= 2 mu g/mL (in both the presence and absence of 50 mu M ZnSO4). To validate the sanitizing activity of compounds 56, 58 and 66, a quantitative suspension disinfection (sanitizer) test was performed. Sanitizing activity (>5log(10) CFU/mL reduction in 5 min) was observed against both S. uberis and E. coli at compound concentrations as low as 1 mg/mL (compounds 56, 58 and 66), and against S. aureus at 1 mg/mL (compound 58); thereby validating the potential of compounds 56, 58 and 66 to function as topical sanitizers designed explicitly for use in non-human applications.

The interest in applications of low-wavenumber Raman (LWR) spectroscopy for pharmaceutical analysis has increased rapidly over the last decade. In this study, a collection of LWR spectra are presented for 75 commonly used pharmaceutical excipients, which serve an important role in the manufacture and delivery of pharmaceutics. The excipients were divided into 10 main groups based on chemical composition: amino acids, mono-and disaccharides, polysaccharides, monomeric polyols, esters, inorganic substances, organosulfates and carboxylic acids/salts, protein, polymers and others. The spectral data is intended to serve as a reference for a number of general purposes such as qualitative and quantitative analysis, counterfeit detection and pharmaceutical process control.

A series of substituted sulfonamide bioisosteres of 8-hydroxyquinoline were evaluated for their antibacterial activity against the common mastitis causative pathogens Streptococcus uberis, Staphylococcus aureus and Escherichia coli, both in the presence and absence of supplementary zinc. Compounds 9a-e, 10a-c, 11a-e, 12 and 13 were demonstrated to have MICs of 0.0625 µg/mL against S. uberis in the presence of 50 µM ZnSO4. Against S. aureus compounds 9g (MIC 4 µg/mL) and 11d (MIC 8 µg/mL) showed the greatest activity, whereas all compounds were found to be inactive against E. coli (MIC > 256 µg/mL); again in the presence of 50 µM ZnSO4. All compounds were demonstrated to be significantly less active in the absence of supplementary zinc. Compound 9g was subsequently confirmed to be bactericidal, with an MBC (≥3log10 cfu/mL reduction) of 0.125 µg/mL against S. uberis in the presence of 50 µM ZnSO4. To validate the sanitising activity of compound 9g in the presence of supplementary zinc, a quantitative suspension disinfection (sanitizer) test was performed. In this preliminary test, sanitizing activity (>5log10 reduction of CFU/mL in 5 min) was observed against S. uberis for compound 9g at concentrations as low as 1 mg/mL, validating the potential of this compound to function as a topical sanitizer against the major environmental mastitis-causing microorganism S. uberis.

The aim of this study is to prepare and characterize an amino-dextran nanoparticle (aDNP) platform and investigate two loading strategies for unmethylated cytosine-phosphate-guanine (CpG) oligonucleotide. aDNP was prepared by desolvation of amino-dextran followed by the chemical crosslinking of amino groups. Size, surface charge, and surface morphology of aDNP was determined by dynamic light scattering and transmission electron microscopy. CpG was either loaded onto aDNP by adsorption (CpG-adsorbed-aDNP) or conjugated to aDNP (CpG-conjugated-aDNP). In vitro cytokine production by bone marrow-derived dendritic cells (BMDCs) was measured by flow cytometry. aDNPs size and zeta potential could be controlled to produce uniform particles in the size range of 50 to 300 nm, surface charge of −16.5 to +14 mV, and were spherical in shape. Formulation control parameters investigated included the anti-solvent, water-to-anti-solvent ratio, level of amine functionality of dextran, and the molar ratio of glutaraldehyde to amine. aDNP could be lyophilized without additional cryoprotectant. Unloaded cationic aDNP (+13 mV) showed acceptable in vitro hemolysis. Unloaded and CpG-loaded aDNPs showed no cytotoxicity on BMDCs. CpG-loaded nanoparticles stimulated cytokine production by BMDCs, the level of cytokine production was higher for CpG-conjugated-aDNP compared to CpG-absorbed-aDNP. aDNP is a promising new drug delivery platform as its offers versatility in loading and tuning of particle properties.

Broccoli sprout powder is a rich source of glucosinolates, which are hydrolysed to isothiocyanates in the presence of the enzyme myrosinase. We showed that in vitro incubation of broccoli sprout powder extract with isolated lymphocytes resulted in the upregulation of transcription factor Nrf2, however, there was no increase in Nrf2 protein levels in lymphocytes isolated 3 h following the ingestion of broccoli sprout powder by healthy volunteers. This highlights the general issue that potential health benefits of food-derived compounds can be compromised by limitations in bioavailability. In vitro experiments showed that the generation of isothiocyanates was reduced when the powder was first exposed to the low pH (1.2) of the stomach and then transferred to the higher pH (6.8) of the intestine. The loss of activity due to pre-exposure to the low stomach pH indicates that formulating the broccoli sprout powder in gastro-resistant formulations should increase that amount of isothiocyanate generated in the intestine for absorption. Gelatin capsules were hand-coated with either Eudragit® L100 or Eudragit® L100-55 and were assessed for their gastro-resistant properties using paracetamol as a model active for dissolution studies. Disintegration and dissolution studies showed that Eudragit® L100-55 coated capsules and DRcapsTM (Capsugel®) failed the United States Pharmacopeia (USP) requirements for gastro-resistant capsules, whereas the Eudragit® L100 coated capsules passed. Five healthy participants were administered 1 g of broccoli sprout powder, ingested either with water or encapsulated in uncoated or gastro-resistant capsules. Urinary excretion of isothiocyanate metabolites over the 24 h period post ingestion was assessed by HPLC. Broccoli sprout powder and uncoated gelatin-encapsulated powder showed comparable excretion of isothiocyanate metabolites (18.4 ± 2.3 and 23.9 ± 2.7 µmol, respectively). The enteric coated capsules provided a significantly longer Tmax than the uncoated gelatin capsules (15.4 ± 2.3 versus 3.7 ± 0.7 h, respectively), indicating protection from disintegration in the stomach, however, the excretion of isothiocyanate metabolites was significantly decreased compared with uncoated capsules (i.e., 8.5 ± 1.1 µmol). The lower in vivo formation or absorption of isothiocyanates observed for the gastro-resistant capsules may be due to participant variation in intestinal pH or transit times, resulting in inappropriate pH conditions or insufficient time for the complete disintegration and dissolution of the capsules.

BACKGROUND:A key challenge in the manufacturing of polymeric colloids is producing nanoparticles with good batch-to-batch consistency.OBJECTIVE:Develop a robust microfluidics method for the preparation of PEG-PLGA nanoparticles using dimethyl sulfoxide (DMSO) as the organic phase solvent for the encapsulation of DMSO soluble agents.METHODS:Microfluidic process parameters, total flow rate (10 mL/min), flow rate ratio (1:1) of the aqueous phase and the organic polymer solution, and polymer concentration (5 mg/ml). Polyvinyl alcohol (PVA) or human serum albumin (HSA) was included in the aqueous phase. Dynamic light scattering and transmission electron microscopy were used to investigate the size and morphology of particles.RESULTS:PLGA nanoparticles made using DMSO with the aqueous solvent containing PVA (2%) had an average size of 60 nm while PLGA-PEG nanoparticles made with and without PVA (2%) had an average size of 70 and 100 nm, respectively. PLGA-PEG nanoparticles generated with or without PVA had a high batch-to-batch coefficient of variation for the particle size of 20% while for PLGA nanoparticles with PVA it was 4%. HSA added to the aqueous phase reduced the size and the zeta potential of PEG-PLGA nanoparticles as well the batch-to-batch coefficient of variation for particle size to < 5%. Nanoparticles were stable in solution and after lyophilized in the presence of sucrose.CONCLUSION:Albumin was involved in the self-assembly of PEG-PLGA nanoparticles altering the physicochemical properties of nanoparticles. Adding protein to the aqueous phase in the microfluidic fabrication process may be a valuable tool for tuning the properties of nanoparticles and improving batch-to-batch consistency.

This article contains the uptake data of two reducible antigen-adjuvant conjugates with different sensitivities to the extracellular and intracellular reductive environment. Using a linker with different redox sensitivity the adjuvant cytosine-phosphate-guanine (CpG) was conjugated to the fluorescently labeled model tumour antigen ovalbumin (OVA). The uptake of the conjugates by dendritic cells in a total splenocyte culture was determined using flow cytometry. The data presented in this paper supports the finding in the research article “Intracellular cleavable CpG oligodeoxynucleotide – antigen conjugate enhances anti-tumour immunity” (Kramer et al., 2016) [1].

Introduction: Virus-like particle (VLP) vaccines face significant challenges in their translation from laboratory models, to routine clinical administration. While some VLP vaccines thrive and are readily adopted into the vaccination schedule, others are restrained by regulatory obstacles, proprietary limitations, or finding their niche amongst the crowded vaccine market. Often the necessity to supplant an existing vaccination regimen possesses an immediate obstacle for the development of a VLP vaccine, despite any preclinical advantages identified over the competition. Novelty, adaptability and formulation compatibility may prove invaluable in helping place VLP vaccines at the forefront of vaccination technology. Areas covered: The purpose of this review is to outline the diversity of VLP vaccines, VLP-specific immune responses, and to explore how modern formulation and delivery techniques can enhance the clinical relevance and overall success of VLP vaccines. Expert commentary: The role of formation science, with an emphasis on the diversity of immune responses induced by VLP, is underrepresented amongst clinical trials for VLP vaccines. Harnessing such diversity, particularly through the use of combinations of select excipients and adjuvants, will be paramount in the development of VLP vaccines.