Covalent isothiocyanate inhibitors of macrophage migration inhibitory factor as potential colorectal cancer treatments.

Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine with roles in innate and adaptive human immune responses, as well as inflammation. MIF exerts its biological activity by binding to the cell surface receptor CD74 as well as intracellular signalling proteins. MIF also possesses keto-enol tautomerase activity. Inhibition of the tautomerase activity has been associated with loss of biological activity of MIF and a potential anticancer target. Isothiocyanates (ITCs) are a class of compounds present in cruciferous vegetables that inhibit the MIF tautomerase activity via covalent modification of the N-terminal proline. A range of substituted ITCs featuring benzyl, phenethyl and phenyl propyl isothiocyanates were designed, synthesised and tested to determine any structure activity relationship for inhibiting MIF. Crystal structures of covalent compounds 8 and 9 in complex with rhMIF revealed key hydrogen bonding and edge-to-face π stacking interactions. Compound 9 and 11 with sub micromolar activity were tested in the NCI60 cancer cell lines panel. Both compounds showed tissue-specific reduced growth in colon and renal cancer cell lines, while one of these showed potent, dose-dependent inhibition of growth against all seven colon cancer cell lines (GI50 < 2.5 µM) and all eight renal cancer cell lines (GI50 < 2.2 µM).

Site-Specific Antimicrobial Activity of a Dual-Responsive Ciprofloxacin Prodrug.

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.

Bioactive atropisomers: Unraveling design strategies and synthetic routes for drug discovery.

Atropisomerism, an expression of axial chirality caused by limited bond rotation, is a prominent aspect within the field of medicinal chemistry. It has been shown that atropisomers of a wide range of compounds, including established FDA-approved drugs and experimental molecules, display markedly different biological activities. The time-dependent reversal of chirality in atropisomers poses complexity and obstacles in the process of drug discovery and development. Nonetheless, recent progress in understanding atropisomerism and enhanced characterization methods have greatly assisted medicinal chemists in the effective development of atropisomeric drug molecules. This article provides a comprehensive review of their special design thoughts, synthetic routes, and biological activities, serving as a reference for the synthesis and biological evaluation of bioactive atropisomers in the future.

Synthesis and Biological Evaluation of Aurachin D Analogues as Inhibitors of Mycobacterium tuberculosis Cytochrome bd Oxidase.

A revised total synthesis of aurachin D (1a), an isoprenoid quinolone alkaloid that targets Mycobacterium tuberculosis (Mtb) cytochrome bd (cyt-bd) oxidase, was accomplished using an oxazoline ring-opening reaction. The ring opening enabled access to a range of electron-poor analogues, while electron-rich analogues could be prepared using the Conrad-Limpach reaction. The aryl-substituted and side-chain-modified aurachin D analogues were screened for inhibition of Mtb cyt-bd oxidase and growth inhibition of Mtb. Nanomolar inhibition of Mtb cyt-bd oxidase was observed for the shorter-chain analogue 1d (citronellyl side chain) and the aryl-substituted analogues 1g/1k (fluoro substituent at C6/C7), 1t/1v (hydroxy substituent at C5/C6) and 1u/1w/1x (methoxy substituent at C5/C6/C7). Aurachin D and the analogues did not inhibit growth of nonpathogenic Mycobacterium smegmatis, but the citronellyl (1d) and 6-fluoro-substituted (1g) inhibitors from the Mtb cyt-bd oxidase assay displayed moderate growth inhibition against pathogenic Mtb (MIC = 4-8 µM).

Development of a bioorthogonal fluorescence-based assay for assessing drug uptake and delivery in bacteria.

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.

Improving Antibacterial Activity of a HtrA Protease Inhibitor JO146 against Helicobacter pylori: A Novel Approach Using Microfluidics-Engineered PLGA Nanoparticles.

Nanoparticle drug delivery systems have emerged as a promising strategy for overcoming limitations of antimicrobial drugs such as stability, bioavailability, and insufficient exposure to the hard-to-reach bacterial drug targets. Although size is a vital colloidal feature of nanoparticles that governs biological interactions, the absence of well-defined size control technology has hampered the investigation of optimal nanoparticle size for targeting bacterial cells. Previously, we identified a lead antichlamydial compound JO146 against the high temperature requirement A (HtrA) protease, a promising antibacterial target involved in protein quality control and virulence. Here, we reveal that JO146 was active against Helicobacter pylori with a minimum bactericidal concentration of 18.8-75.2 µg/mL. Microfluidic technology using a design of experiments approach was utilized to formulate JO146-loaded poly(lactic-co-glycolic) acid nanoparticles and explore the effect of the nanoparticle size on drug delivery. JO146-loaded nanoparticles of three different sizes (90, 150, and 220 nm) were formulated with uniform particle size distribution and drug encapsulation efficiency of up to 25%. In in vitro microdilution inhibition assays, 90 nm nanoparticles improved the minimum bactericidal concentration of JO146 two-fold against H. pylori compared to the free drug alone, highlighting that controlled engineering of nanoparticle size is important in drug delivery optimization.

Design, synthesis and biological evaluation of P2-modified proline analogues targeting the HtrA serine protease in Chlamydia.

High temperature requirement A (HtrA) serine proteases have emerged as a novel class of antibacterial target, which are crucial in protein quality control and are involved in the pathogenesis of a wide array of bacterial infections. Previously, we demonstrated that HtrA in Chlamydia is essential for bacterial survival, replication and virulence. Here, we report a new series of proline (P2)-modified inhibitors of Chlamydia trachomatis HtrA (CtHtrA) developed by proline ring expansion and Cγ-substitutions. The structure-based drug optimization process was guided by molecular modelling and in vitro pharmacological evaluation of inhibitory potency, selectivity and cytotoxicity. Compound 25 from the first-generation 4-substituted proline analogues increased antiCtHtrA potency and selectivity over human neutrophil elastase (HNE) by approximately 6- and 12-fold, respectively, relative to the peptidic lead compound 1. Based on this compound, second-generation substituted proline residues containing 1,2,3-triazole moieties were synthesized by regioselective azide-alkyne click chemistry. Compound 49 demonstrated significantly improved antichlamydial activity in whole cell assays, diminishing the bacterial infectious progeny below the detection limit at the lowest dose tested. Compound 49 resulted in approximately 9- and 22-fold improvement in the inhibitory potency and selectivity relative to 1, respectively. To date, compound 49 is the most potent HtrA inhibitor developed against Chlamydia spp.

SMA-BmobaSNO: an intelligent photoresponsive nitric oxide releasing polymer for drug nanoencapsulation and targeted delivery.

Nitric oxide (NO) is an important biological signalling molecule that acts to vasodilate blood vessels and change the permeability of the blood vessel wall. Due to these cardiovascular actions, co-administering NO with a therapeutic could enhance drug uptake. However current NO donors are not suitable for targeted drug delivery as they systemically release NO. To overcome this limitation we report the development of a smart polymer, SMA-BmobaSNO, designed to release NO in response to a photostimulus. The polymer's NO releasing functionality is an S-nitrosothiol group that, at 10 mg ml-1, is highly resistant to both thermal (t1/216 d) and metabolic (t1/232 h) decomposition, but rapidly brakes down under photoactivation (2700 W m-2, halogen source) to release NO (t1/225 min). Photoresponsive NO release from SMA-BmobaSNO was confirmed in a cardiovascular preparation, where irradiation resulted in a 12-fold decrease in vasorelaxation EC50(from 5.2µM to 420 nM). To demonstrate the polymer's utility for drug delivery we then used SMA-BmobaSNO to fabricate a nanoparticle containing the probe Nile Red (NR). The resulting SMA-BmobaSNO-NR nanoparticle exhibited spherical morphology (180 nm diameter) and sustained NR release (≈20% over 5 d). Targeted delivery was characterised in an abdominal preparation, where photoactivation (450 W m-2) caused localized increases in vasodilation and blood vessel permeability, resulting in a 3-fold increase in NR uptake into photoactivated tissue. Nanoparticles fabricated from SMA-BmobaSNO therefore display highly photoresponsive NO release and can apply the Trojan Horse paradigm by using endogenous NO signalling pathways to smuggle a therapeutic cargo into target tissue.

Hydrogen Sulfide-Responsive Bicontinuous Nanospheres.

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 150-200 nm 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.

EGFR-targeted prodrug activation using bioorthogonal alkene-azide click-and-release chemistry.

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 proof-of-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.

Optimization of peptide-based inhibitors targeting the HtrA serine protease in Chlamydia: Design, synthesis and biological evaluation of pyridone-based and N-Capping group-modified analogues.

The obligate intracellular bacterium Chlamydia trachomatis (C. trachomatis) is responsible for the most common bacterial sexually transmitted infection and is the leading cause of preventable blindness, representing a major global health burden. While C. trachomatis infection is currently treatable with broad-spectrum antibiotics, there would be many benefits of a chlamydia-specific therapy. Previously, we have identified a small-molecule lead compound JO146 [Boc-Val-Pro-ValP(OPh)2] targeting the bacterial serine protease HtrA, which is essential in bacterial replication, virulence and survival, particularly under stress conditions. JO146 is highly efficacious in attenuating infectivity of both human (C. trachomatis) as well as koala (C. pecorum) species in vitro and in vivo, without host cell toxicity. Herein, we present our continuing efforts on optimizing JO146 by modifying the N-capping group as well as replacing the parent peptide structure with the 2-pyridone scaffold at P3/P2. The drug optimization process was guided by molecular modelling, enzyme and cell-based assays. Compound 18b from the pyridone series showed improved inhibitory activity against CtHtrA by 5-fold and selectivity over human neutrophil elastase (HNE) by 109-fold compared to JO146, indicating that 2-pyridone is a suitable bioisostere of the P3/P2 amide/proline for developing CtHtrA inhibitors. Most pyridone-based inhibitors showed superior anti-chlamydial potency to JO146 especially at lower doses (25 and 50 µM) in C. trachomatis and C. pecorum cell culture assays. Modifications of the N-capping group of the peptidyl inhibitors did not have much influence on the anti-chlamydial activities, providing opportunities for more versatile alterations and future optimization. In summary, we present 2-pyridone based analogues as a new generation of non-peptidic CtHtrA inhibitors, which hold better promise as anti-chlamydial drug candidates.

Self-immolative Linkers in Prodrugs and Antibody Drug Conjugates in Cancer Treatment.

BACKGROUND: The design of anti-cancer therapies with high anti-tumour efficacy and reduced toxicity continues to be challenging. Anti-cancer prodrug and antibody-drug-conjugate (ADC) strategies that can specifically and efficiently deliver cytotoxic compounds to cancer cells have been used to overcome some of the challenges. The key to the success of many of these strategies is a self-immolative linker, which after activation can release the drug payload. Various types of triggerable self-immolative linkers are used in prodrugs and ADCs to improve their efficacy and safety. OBJECTIVE: Numerous patents have reported the significance of self-immolative linkers in prodrugs and ADCs in cancer treatment. Based on the recent patent literature, we summarise methods for designing the site-specific activation of non-toxic prodrugs and ADCs in order to improve selectivity for killing cancer cells. METHODS: In this review, an integrated view of the potential use of prodrugs and ADCs in cancer treatment are provided. This review presents recent patents and related publications over the past ten years uptill 2020. RESULTS: The recent patent literature has been summarised for a wide variety of self-immolative PABC linkers, which are cleaved by factors including responding to the difference between the extracellular and intracellular environments (pH, ROS, glutathione) through over-expressed enzymes (cathepsin, plasmin, ß-glucuronidase) or bioorthogonal activation. The mechanism for self-immolation involves the linker undergoing a 1,4- or 1,6-elimination (via electron cascade) or intramolecular cyclisation to release cytotoxic drug at the targeted site. CONCLUSION: This review provides the commonly used strategies from recent patent literature in the development of prodrugs based on targeted cancer therapy and antibody-drug conjugates, which show promise in therapeutic applications.

Synthesis and Biological Evaluation of (-) and (+)-Spiroleucettadine and Analogues.

A second-generation enantiospecific synthesis of spiroleucettadine is described. The original reported antibacterial activity was not observed when the experiment was repeated on the synthetic samples; however, significant anti-proliferative activity was uncovered for both enantiomers of spiroleucettadine. Comparison of the optical rotational data and ORD-CD spectra of both enantiomers and the reported spectrum from the natural source have not provided a definitive answer regarding the absolute stereochemistry of naturally occurring spiroleucettadine. Efforts then focussed on alteration at the C-4 and C-5 positions of the slightly more active (-)-spiroleucettadine. Ten analogues were synthesised, with three analogues found to possess similar anti-proliferative profiles to spiroleucettadine against the H522 lung cancer cell line.

Tuning activation and self-immolative properties of the bioorthogonal alkene-azide click-and-release strategy.

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.

Tetrafluoroaryl azide as an N-terminal capping group for click-to-dissolve diphenylalanine hydrogels.

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.

Controlled Delivery of Nitric Oxide for Cancer Therapy.

Nitric oxide (NO) is a short-lived, endogenously produced, signaling molecule which plays multiple roles in mammalian physiology. Underproduction of NO is associated with several pathological processes; hence a broad range of NO donors have emerged as potential therapeutics for cardiovascular and respiratory disorders, wound healing, the immune response to infection, and cancer. However, short half-lives, chemical reactivity, rapid systemic clearance, and cytotoxicity have hindered the clinical development of most low molecular weight NO donors. Hence, for controlled NO delivery, there has been extensive effort to design novel NO-releasing biomaterials for tumor targeting. This review covers the effects of NO in cancer biology, NO releasing moieties which can be used for NO delivery, and current advances in the design of NO releasing biomaterials focusing on their applications for tumor therapy.

Structure-activity analysis of peptidic Chlamydia HtrA inhibitors.

Chlamydia trachomatis high temperature requirement A (CtHtrA) is a serine protease that performs proteolytic and chaperone functions in pathogenic Chlamydiae; and is seen as a prospective drug target. This study details the strategies employed in optimizing the irreversible CtHtrA inhibitor JO146 [Boc-Val-Pro-ValP(OPh)2] for potency and selectivity. A series of adaptations both at the warhead and specificity residues P1 and P3 yielded 23 analogues, which were tested in human neutrophil elastase (HNE) and CtHtrA enzyme assays as well as Chlamydia cell culture assays. Trypsin and chymotrypsin inhibition assays were also conducted to measure off-target selectivity. Replacing the phosphonate moiety with α-ketobenzothiazole produced a reversible analogue with considerable CtHtrA inhibition and cell culture activity. Tertiary leucine at P3 (8a) yielded approximately 33-fold increase in CtHtrA inhibitory activity, with an IC50 = 0.68 ±â€¯0.02 µM against HNE, while valine at P1 retained the best anti-chlamydial activity. This study provides a pathway for obtaining clinically relevant inhibitors.

Alkene-Azide 1,3-Dipolar Cycloaddition as a Trigger for Ultrashort Peptide Hydrogel Dissolution.

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 10 h, 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.

Honey reduces the metastatic characteristics of prostate cancer cell lines by promoting a loss of adhesion.

Honey has been shown to have a range of therapeutic effects in humans, with anti-inflammatory and anti-bacterial effects among those previously characterised. Here, we examine the possibility of New Zealand thyme, manuka and honeydew honeys, and their major sugar and phenolic components, reducing the development of metastatic cancer. Their activity was examined in vitro, in PC3 and DU145 prostate cancer cell lines, through measuring the compounds' effects on the metastatic characteristics of migration, invasion and adhesion. First, the phenolic compounds gallic acid, caffeic acid, quercetin, kaempferol and chrysin were quantified in the honeys using high performance liquid chromatography, and found in nanomolar concentrations. In a Boyden chamber-based migration assay, non-toxic concentrations of thyme and honeydew honeys reduced cell migration by 20%, and all phenolic compounds except caffeic acid also lowered migration, although a mixture of only the sugars found in honey had no effect. All of the honeys, phenolics and the sugar-only mixture reduced invasive movement of cells through extracellular matrix by up to 75%. Most notably, each of the three honeys and the sugar-only mixture reduced cell adhesion to collagen I by 90%. With the exception of quercetin, phenolic compounds did not reduce adhesion. Therefore, honey and its sugar and phenolic components can lower the metastatic properties of cancer cells, and may do this by preventing effective cell adhesion to the extracellular matrix. The sugars and phenol compounds of honey are much more effective in combination than individually.

Poloxamer 407-chitosan grafted thermoresponsive hydrogels achieve synchronous and sustained release of antigen and adjuvant from single-shot vaccines.

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.