Chemoresistance is a major obstacle to effective cancer treatment, particularly in tubo-ovarian high-grade serous carcinoma (HGSC), the most lethal gynaecological malignancy. Predicting patient-specific chemoresistance remains challenging due to tumour heterogeneity and the lack of reliable biomarkers. Raman spectroscopy, a label-free technique that provides biochemical insights into cells and tissues without the need for specific biomarkers, has been extensively applied in cancer research, but its full potential for detecting subtle biochemical changes linked to chemoresistance in HGSC at the single-cell and subcellular levels remains underexplored. Another critical challenge is the estimation of classification performance on future data with cross-validation (CV) in the presence of batch effects. In this study, we demonstrated that confocal Raman microscopy combined with multivariate analysis can discriminate between cisplatin-resistant (TYK-nu-CP.r) and cisplatin-sensitive (TYK-nu) HGSC cell lines with 78% accuracy without batch correction. After batch correction, the accuracy improved to 84%. Feature importance analysis suggested that the separation was linked to a higher level of lipid unsaturation and elevated glutathione levels in the chemosensitive cell line. Additionally, we proposed a new CV-based area under the receiver operating characteristic curve (AUC) estimator that accounts for the batch effects better than the popularly used leave-one-batch-out CV. Together, these results show that with careful data processing, accounting for biases and batch effects, Raman microscopy enables reliable detection of chemoresistance at a cellular level and can provide insights into the molecular basis of chemoresistance. This study suggests that Raman microscopy holds promise as a tool for predicting chemoresistance in HGSC and guiding personalised treatment strategies.

Hydrogen sulfide (H2S) imbalance has been implicated in pathologies, and reinstating H2S homeostasis could be a useful therapeutic strategy. However, delivery of H2S to the disease site remains a challenge. Functionalised nanoformulations could be used as a strategy to deliver high concentrations of H2S in a targeted manner. Use of a disease-associated trigger that activates and releases H2S would provide therapeutic selectivity. As proof-of-concept, synthesis and formulation of block co-polymers bearing a thiocarbamate bond, a carbonyl sulfide (COS) precursor, is described. Activation by hydrogen peroxide (H2O2), and a subsequent 1,6-self-immolation process leads to release of COS, which in the presence of carbonic anhydrase is hydrolysed to H2S. H2S generation was exemplified by reduction of an azido-pro-fluorophore. Formulation of the polymer resulted in compound vesicles that were able to encapsulate a model drug and could be useful in future biological studies exploring delivery of H2S as a therapeutic, or to activate azido-masked prodrug/pro-fluorophore in areas of high reactive oxygen species (ROS).

Bioorthogonal click-to-release prodrug activation strategies should be fast and high yielding. However, in strain-promoted alkene-azide click-to-release, a fast click-step translates to slow and inefficient drug release. To improve drug yield, branched dual-core scaffolds that increase drug release are reported. The dual-core consists of an exposed electron-deficient aryl azide (core-1) attached to an electron-rich self-immolating branched linker (core-2). Core-1, a tetrafluoroaryl azide with methyl-substitution at the benzylic carbon, facilitates a rapid click reaction with trans-cyclooctenes (TCOs). Core-2 had an N-methyl carbamate or ether linker and a 1,4/1,4- or 1,4/1,6-self-immolating scaffold. Various release rates of a fluorophore were observed across the series, with 1,4/1,6-self-immolating core-2, linked via the N-methyl carbamate, having sustained and enhanced fluorophore release. The second-order rate constant for cycloaddition of this 1,4/1,6-self-immolating analogue and d-TCO is, to the best of our knowledge, the fastest to date (22.0 M-1 s-1). A persistent intermediate was observed, leading to a long, sustained release of fluorophore from the branched scaffold, though activation under acidic conditions (pH = 5.5) led to ≈ 15% higher fluorophore release compared to activation at pH 7.4. Therefore, combinations of core-1 and core-2 could improve future pretargeted in vivo bioorthogonal click-to-release prodrug strategies, especially in the acidic tumor environment.

Bacterial infections create distinctive microenvironments with a unique mix of metabolites and enzymes compared with healthy tissues that can be used to trigger the activation of antibiotic prodrugs. Here, a single and dual prodrug masking the C3 carboxylate and C7 piperazine of the fluoroquinolone, ciprofloxacin, responsive to nitroreductase (NTR) and/or hydrogen sulfide (H2S), was developed. Masking both functional groups reduced the activity of the prodrug against Staphylococcus aureus and Escherichia coli, increasing its minimum inhibitory concentration (MIC) by ∼512-fold (S. aureus) and ∼8000-fold (E. coli strains), while masking a single group only increased the MIC by ∼128-fold. Bacteria subjected to prolonged prodrug exposure did not show any increase in resistance. Triggering assays demonstrated the conversion of prodrugs to ciprofloxacin, and in a murine infection model, responsive prodrugs showed antibacterial activity comparable to that of ciprofloxacin, suggesting in vivo activation of prodrugs. Thus, the potential for site-specific antibiotic treatment with reduced threat of resistance is demonstrated.

Bioorthogonal chemistry can facilitate the development of fluorescent probes that can be used to sensitively and specifically detect the presence of biological targets. In this study, such an assay was developed to evaluate the uptake and delivery of antimicrobials into Escherichia coli, building on and extending previous work which utilised more resource intensive LCMS detection. The bacteria were genetically engineered to express streptavidin in the periplasmic or cytoplasmic compartments, which was used to localise a bioorthogonal probe (BCN-biotin). Azido-compounds which are delivered to these compartments react with the localised BCN-biotin-streptavidin in a concentration-dependent manner via a strain-promoted alkyne-azide cycloaddition. The amount of azido-compound taken up by bacteria was determined by quantifying unreacted BCN-biotin-streptavidin via an inverse electron demand Diels-Alder reaction between remaining BCN-biotin and a tetrazine-containing fluorescent dye. Following optimisation and validation, the assay was used to assess uptake of liposome-formulated azide-functionalised luciferin and cefoxitin. The results demonstrated that formulation into cationic liposomes improved the uptake of azide-functionalised compounds into the periplasm of E. coli, providing insight on the uptake mechanism of liposomes in the bacteria. This newly developed bioorthogonal fluorescence plate-reader based assay provides a bioactivity-independent, medium-to-high throughput tool for screening compound uptake/delivery.

Cancer vaccines have been developed as an additional method of treatment in the fight against cancer. However, an important barrier to an effective vaccine is the inefficient presentation of exogenous antigen by dendritic cells to cytotoxic CD8 T cells. In this study, DPPC liposomes were modified with channels and loaded with polyethyleneimine (PEI) and 5,6-dimethylxanthenone-4-acetic acid (DMXAA) to produce a vaccine carrier. The liposomes were designed to be pH responsive to facilitate delivery of antigens directly to the cytoplasm of antigen presenting cells, bypassing the cross-presentation pathway and improving cellular immune responses. The lysis of liposomes in acidic cell-free conditions was measured using a validated dynamic light scattering assay in order to gain an insight into the mechanism of PEI-mediated lysis. Dendritic cell stimulation and T cell proliferation was investigated in vitro and the potential of this formulation to stimulate a therapeutic anti-cancer immune response was examined in a murine melanoma model. The modified formulation stimulated T cell activation in vitro and induced a small but significant increase in survival in immunized mice. Overall, liposomes modified with PEI and channels successfully delivered antigen to the cytoplasm of dendritic cells, which subsequently led to the development of an appropriate immune response.

Block copolymers (BCPs) that can self-assemble into particles and be triggered by disease-specific molecules such as hydrogen sulfide (H2S) have the potential to impact on drug delivery, decreasing off-target toxicities while increasing drug efficacy. However, the incorporation of H2S-responsive aryl azides into BCPs for self-assembly has been limited by heat, light, and radical sensitivities. In this study, a robust activator regenerated by the electron-transfer atom-transfer radical polymerization reaction was used to synthesize aryl-azide-containing BCPs under ambient conditions. Conditions controlling self-assembly of the BCPs into 150–200nm particles and the physicochemical properties of the particles were investigated. The use of nanoprecipitation with tetrahydrofuran to promote self-assembly of the BCPs resulted in vesicle structures, while dimethylformamide or dimethylsulfoxide resulted in polymeric bicontinuous nanospheres (BCNs). Triggering of the BCPs and particles (vesicles or BCNs) via exposure to H2S revealed that unsubstituted aryl azides were readily reduced (by HS–), resulting in particle disruption or cross-linking. The relative polar nature of the particle bilayers containing unsubstituted aryl azides and the open structure of the BCNs did however limit encapsulation of small hydrophilic and hydrophobic payloads. Incorporation of a benzylamide substituent onto the aryl azide group increased the hydrophobicity of the particles and encapsulation of hydrophilic cargo but reduced sensitivity to H2S, likely due to the reduced penetration of HS– into the bilayer.

Bacteria biohybrid-based vaccine delivery systems, which integrate a vaccine carrier with live non-pathogenic bacteria, are hypothesized to have improved immunostimulating potential. The aim of this study was to develop oral bacteria biohybrid-based vaccines to treat a mouse model of colorectal cancer. E. coli were combined with tumor antigen- and adjuvant-containing emulsions or liposomes. Emulsion and liposome biohybrid vaccines demonstrated in vitro and in vivo therapeutic potential. Bacteria biohybrid vaccines significantly increased the expression of CD40+, CD80+ and CD86+ on murine bone marrow-derived dendritic cells. Mice vaccinated with emulsion biohybrid vaccines had an increased CD8+ T cell infiltration into tumors and developed three-fold smaller tumors compared to the mice that received emulsion vaccine without E. coli.

The potential of cubosomes to improve delivery of incorporated cargo to the brain was explored in zebrafish. Cubosomes were formulated with one of three stabilisers, Pluronic F68, Pluronic F127 or Tween 80, with the hypothesis that coating with Tween 80 will enable brain targeting of cubosomes as has been previously shown for polymeric nanoparticles. The physiochemical properties and the ability of the cubosomes to facilitate delivery of the model drug lissamine rhodamine (RhoB) into the brain was investigated. Distribution of cubosomes in the midbrain was also investigated by ultrastructural analysis via incorporation of octanethiol-functionalized gold nanoparticles. Cubosomes were typically 165-195 nm in size with a Pn3m (Pluronics) or Im3m (Tween 80) cubic phase internal structure. Cubosomes were injected intravenously into zebrafish larvae (12-14 days post fertilization) and the concentration of RhoB in the midbrain was determined by quantifying its fluorescence intensity. Uptake of RhoB was significantly greater in larvae injected with Tween 80 stabilized cubosomes as compared to a control suspension of RhoB or cubosomes stabilized with Pluronics. Collectively, we show for the first time that cubosomes can be functionalized to deliver drug across the BBB, offering new opportunities to overcome drug delivery issues across this formidable biological barrier.

Epidermal growth factor receptor (EGFR) is overexpressed in many cancers and therefore serves as an excellent target for prodrug activation. Functionalised trans-cyclooctenes (TCO) were conjugated to an EGFR antibody (cetuximab), providing a reagent for pre-targeting and localisation of the bioorthogonal reagent. The TCOs react with a 4-azidobenzyl carbamate doxorubicin prodrug via a [3 + 2]-cycloaddition and subsequent self-immolation leads to release of doxorubicin (click-and-release). In vitro cell-based assays demonstrated proofof-concept, that cetuximab conjugated to highly strained TCO (AB-d-TCO) could bind to the EGFR in a melanoma cell line, and selectively activate the doxorubicin prodrug. In a non-EGFR expressing melanoma cell line, no significant prodrug activation was observed. In vivo experiments using this combination of AB-d-TCO and the azido-doxorubicin prodrug in a murine melanoma model revealed no significant anti-tumour activity or increased survival, suggesting there was insufficient prodrug activation and drug release at the tumour site.

Self-immolative aryl azides can react with trans-cyclooctenes (TCO), triphenylphosphines or hydrogen sulfide (H2S) to activate prodrugs, imaging probes and drug delivery systems. To date, the synthesis of polymers containing these aryl azide self-immolative linkers and their reactivity with a strained alkene (i.e., in a bioorthogonal reaction) has not been explored. Also, due to the instability of aryl azides towards light and high temperatures, the polymerization methods compatible with aryl azides are limited. Through systematic investigation of the reversible addition-fragmentation chain transfer (RAFT) and atom transfer radical polymerization (ATRP) methods, a self-immolative PEG-aryl azide block copolymer (PEG(45)-b-ABOC(28) 2) and a non-responsive 4-fluoroaryl block copolymer (PEG(45)-b-FBOC24 3) was prepared. ATRP provided the desired polymers in a highly controlled manner, whereas the RAFT conditions led to higher levels of aryl azide polymer degradation. The ATRP derived polymers 2 and 3 were formulated into nanoparticles of approximately 200 nm diameter, and particle triggering was demonstrated by the [3+2]-cycloaddition reaction of TCO with PEG(45)-b-ABOC(28) 2 in solution (pure polymer) and as a formulated nanoparticle. Preliminary in vitro cell viability studies suggested that the stimuli-responsive aryl azide polymers/nanoparticles are not cytotoxic up to 200 mu g/ml concentrations.

Implants have long been used in the field of drug delivery as controlled release vehicles and are now being investigated as single-shot vaccine technologies. Implants have shown great promise, minimizing the need for multiple immunizations while stimulating potent immune responses with reduced doses of vaccine. Synchronous release of vaccine components from implants over an appropriate period of time is important in order to avoid issues including immune tolerance, sequestration or deletion. Traditionally, implants require surgical implantation and removal, which can be a barrier to their widespread use. Degradable and in situ implants are now being developed that can be administered using minimally invasive subcutaneous or intramuscular injection techniques. Injectable hydrogels remain the most commonly studied approach for sustained vaccine delivery due to their ease of administration and tunable degradation properties. Despite exciting advancements in the field of vaccine implants, few technologies have progressed to clinical trials. To increase the likelihood of clinical translation of vaccine implants, strategic testing of disease-relevant antigens in appropriate species is essential. In this review, the significance of vaccine implants and the different types of implants being developed to deliver vaccines are discussed.

Implants have long been used in the field of drug delivery as controlled release vehicles and are now being investigated as single-shot vaccine technologies. Implants have shown great promise, minimizing the need for multiple immunizations while stimulating potent immune responses with reduced doses of vaccine. Synchronous release of vaccine components from implants over an appropriate period of time is important in order to avoid issues including immune tolerance, sequestration or deletion. Traditionally, implants require surgical implantation and removal, which can be a barrier to their widespread use. Degradable and in situ implants are now being developed that can be administered using minimally invasive subcutaneous or intramuscular injection techniques. Injectable hydrogels remain the most commonly studied approach for sustained vaccine delivery due to their ease of administration and tunable degradation properties. Despite exciting advancements in the field of vaccine implants, few technologies have progressed to clinical trials. To increase the likelihood of clinical translation of vaccine implants, strategic testing of disease-relevant antigens in appropriate species is essential. In this review, the significance of vaccine implants and the different types of implants being developed to deliver vaccines are discussed.

The aim of this study was to develop an oral vaccine that could be used to treat colorectal cancer. Oral vaccines are technically challenging to develop due to the harsh gastric environment but have numerous benefits including high patient acceptability and the potential to stimulate local mucosal immune responses. Therapeutic vaccines are being investigated as options to treat cancer and the generation of local mucosal immunity may be of benefit in the treatment of gastrointestinal cancers. Novel oral vaccines consisting of a long tumour peptide and the TLR2 (Toll-like receptor 2) ligand Pam2Cys, formulated in either liposomes or W/O/W double emulsions, were developed. Oral dosing with the emulsion vaccine increased the numbers of activated T cells, B cells and CD11c+F4/80+CD11b+ cells compared to mice that received control vaccines. In an orthotopic mouse model of colorectal cancer these immunological changes were associated with a seven-fold reduction in tumour size.

We report on a series of 4-azidobenzyloxy-substituted self-immolative linkers which undergo [3 + 2]-cycloaddition (click reaction) with functionalized trans-cyclooctenes (TCOs) at second-order rate constants in the range of 0.017 to 4.9 M-1 s-1. The choice of 4-azidobenzyloxy-substituted linker and the TCO play a critical role in the rate of all click-and-release steps, which includes the [3 + 2]-cycloaddition and subsequent degradation pathway of the triazoline to an aniline that undergoes 1,6- or 1,8-self-immolation of the phenol. We demonstrate that reacting a 4-azido-2,3,5,6-tetrafluorobenzyloxy-linker with a highly strained TCO (d-TCO) gives, to the best of our knowledge, the fastest TCO-strained alkene-azide click reaction to date (4.9 M-1 s-1), but with one caveat; release of phenol via 1,6-self-immolation is extremely slow. A methyl substituent attached to the benzyl carbon of this analogue maintains the rapid click-reaction rate, but has the added benefit of enabling the release of the phenol within hours. In an aqueous solvent at reagent concentrations in the micromolar range a maximium release was observed after 48 hours; ≈65 and ≈78% of phenol released depending on the TCO used. The new suite of linkers and their combination with TCOs of varying structure add to the toolbox of bioorthogonal click-and-release reactions.

The synthesis of a bioorthogonal-responsive low molecular weight diphenylalanine (PhePhe)-based hydrogel that is capped with a 4-azido-2,3,5,6-tetrafluorobenzyl carbamate self-immolative linker is reported. The hydrogelator (AzF4-PhePhe) generates a stable hydrogel at 0.1 wt%, and rapidly reacts with the bioorthogonal reagent trans-cyclooctene (TCO), inducing a gel-to-solution transition. The critical gel concentration is five-fold lower than our previously synthesized non-fluorinated hydrogelator (Az-PhePhe), and the minimum concentration of TCO required for visible gel-to-solution transition in 24 hours is 1 mM. Doxorubicin can be encapsulated in the hydrogel and TCO-triggered dissolution results in 76% and 89% release after 10 and 24 hours, respectively. Compared with our non-substituted aryl azide capping group used for Az-PhePhe, the tetrafluorinated aryl azide group improves the stability of the hydrogel in unbuffered water at a lower critical gel concentration, while improving sensitivity towards the bioorthogonal reagent TCO.

OBJECTIVE:To compare the characteristics of rutin-loaded PLGA (poly(lactic-coglycolic acid)) nanoparticles prepared using a single emulsion evaporation method (bulk method) and a nanoprecipitation method using microfluidics.METHODS:Rutin-loaded PLGA nanoparticles were produced using different methods and characterized for size, zeta potential, entrapment efficiency (EE) and drug loading (DL). A design of experiments approach was used to identify the effect of method parameters to optimize the formulation. DSC was used to investigate the solid-state characteristics of rutin and PLGA and identify any interactions in the rutin-loaded PLGA nanoparticles. The release of rutin from PLGA nanoparticles was examined in biorelevant media and phosphate buffer (PBS).RESULTS:The optimal formulation of rutin-loaded PLGA nanoparticles produced using a microfluidics method resulted in a higher entrapment efficiency of 34 ± 2% and a smaller size of 123 ± 4 nm compared to a bulk method (EE 27 ± 1%, size 179 ± 13 nm). The solidstate of rutin and PLGA changed from crystalline to amorphous with the preparation of rutin- loaded PLGA nanoparticles. More importantly, using microfluidics, rutin released faster from rutin-loaded PLGA nanoparticles in biorelevant media and PBS with higher burst release compared to the rutin release from the nanoparticles prepared by using the bulk method.CONCLUSION:Rutin can be encapsulated in nanoparticles formulated with different methods with mean sizes of less than 200 nm. Microfluidics produced more uniform rutin-loaded PLGA nanoparticles with a higher EE, DL and faster release compared to a bulk production method.

Sustained-release vaccine delivery systems may enhance the immunogenicity of subunit vaccines and reduce the need for multiple vaccinations. The aim of this study was to develop a thermoresponsive hydrogel using poloxamer 407-chitosan (CP) grafted copolymer as a delivery system for single-shot sustained-release vaccines. The CP copolymer was synthesized using 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide and N-hydroxysuccinimide chemistry. The CP copolymer was a free flowing solution at ambient temperature and transformed rapidly into a gel at body temperature. The hydrogels were loaded with vaccine antigen and adjuvants or the vaccine components were encapsulated in poly (lactic-co-glycolic acid) nanoparticles in order to ensure synchronous release. The CP hydrogels were stable for up to 18 days in vitro. Release of both nanoparticles and the individual components was complete, with release of the individual components being modulated by incorporation into nanoparticles. In vivo, a single dose of CP hydrogel vaccine induced strong, long lasting, cellular and humoral responses that could protect against the development of tumors in a murine melanoma model.

An alkene-azide 1,3-dipolar cycloaddition between trans-cyclooctene (TCO) and an azide-capped hydrogel that promotes rapid gel dissolution is reported. Using an ultrashort aryl azide-capped peptide hydrogel (PhePhe), we have demonstrated proof-of-concept where upon reaction with TCO, the hydrogel undergoes a gel-sol transition via 1,2,3-triazoline degradation and 1,6-self-immolation of the generated aniline. The potential application of this as a general trigger in sustained drug delivery is demonstrated through release of encapsulated cargo (doxorubicin). Administration of TCO resulted in 87% of the cargo being released in 10h, compared to 13-14% in the control gels. This is the first example of a potential bioorthogonal-triggered hydrogel dissolution using a traditional click-type reaction. This type of stimulus could be extended to other aryl azide-capped hydrogels.

Subunit vaccine formulations are often produced as liquid dispersions through complicated processes. It is desirable, however, to have simple, cheap and up-scalable methods to produce nanoparticulate subunit vaccines in powder form. Here, a simple single-step spray drying process for production of powder cubosome precursors with the model antigen ovalbumin (OVA) and the adjuvant Quil-A is presented. The cubosomes were characterized in vitro and evaluated in vivo by subcutaneous and oral administration for their potential as a vaccine formulation. Hydrated cubosomes had average particle size of 257 +/- 8 nm and zeta potential of -18.0 +/- 0.6 mV. The powder contained 10.6 +/- 0.7% w/w OVA prior to hydration, of which 65 +/- 1% was released within the first 20 min in 9.5 mM PBS at pH 7.3, with the remaining OVA gradually released over the following 24 h. Immunization with cubosomes resulted in significantly stronger antigen-specific serum IgG responses (p < 0.01), CD8(+) T cell expansion (p < 0.0001) and target T cell killing compared to controls when given s.c., and was ineffective orally. This study shows that spray drying is a suitable method for producing nanoparticulate vaccine formulations in dry powder form.