Gel Depot Research Articles

Endogenous Calcium-Supported dynamic Copper(II)-Crosslinked Gel depot for in situ disulfiram activation and synergistic cancer therapy

The development of smart therapeutic scaffolds to improve chemotherapeutic efficacy and enhance antitumor immunity remains a considerable challenge. In this study, we engineered a therapeutic copper ion-crosslinked gel depot based on alginate to enhance disulfiram (DSF)-mediated cancer chemoimmunotherapy. This gel depot maintained structural stability through ion coordination exchange with the calcium ions in body fluids and continuously created an antitumor diethyldithiocarbamate-copper complex (CuET), which led to the sustained release of various therapeutic agents in vivo. The generated CuET not only had a direct antitumor effect due to its cytotoxicity but also effectively induced the immunogenic death of cancer cells. These factors coregulated the immunosuppressed tumor microenvironment with the released DSF, thus sensitizing the systemic immune response and improving the efficacy of anti-programmed cell death-ligand 1 antibody (aPD-L1)-mediated immune checkpoint blockade. The results indicated that this endogenous calcium-supported therapeutic copper(II)-crosslinked gel depot could effectively improve the tumor immune microenvironment, leading to enhanced synergistic antitumor efficacy in vivo.

Injectable hydrogel imbibed with camptothecin-loaded mesoporous silica nanoparticles as an implantable sustained delivery depot for cancer therapy

In recent years, injectable stimuli-sensitive hydrogels are employed as suitable drug delivery carriers for the release of various anti-cancer drugs. However, large pore size of the microporous hydrogel trigger release of small molecular anticancer drug that limits hydrogel application in cancer therapy. Therefore, introducing reinforcing fillers such as mesoporous silica nanoparticles (MSNs) can not only load different type of anticancer drugs but also prevent the premature release of drugs due to the strengthening of the networks. Furthermore, high specific surface area, suitable size, large pore volume, and stable physicochemical properties of MSNs can improve the therapeutic efficacy. In this study, to sustain the release of hydrophobic anticancer drug, camptothecin (CPT) was loaded into MSNs, and then imbibed into the physiological stimuli-sensitive poly(ethylene glycol)-poly(β-aminoester urethane) (PAEU) hydrogels. MSN-imbibed PAEU hydrogels exhibited prolonged release of CPT than MSNs and PAEU hydrogel alone. Furthermore, MSN-imbibed PAEU copolymers form stable viscoelastic gel depot into the subcutaneous layers of Sprague-Dawley rats and found to be safe and not induced toxicity to healthy organs, implying biodegradability and safety of the hydrogels. Interestingly, CPT-loaded hydrogels shown dose-dependent toxicity to A549 and B16F10 cells. These results demonstrated that MSN-imbibed PAEU hydrogel with biocompatible, biodegradable, and in situ gel forming property could be a useful drug delivery depot for sustained release of anticancer drugs.

Thermosensitive acetylated carboxymethyl chitosan gel depot systems sustained release caffeic acid phenethyl ester for periodontitis treatment

AbstractPeriodontitis is a chronic inflammatory disease of tooth support tissues leading to progressive destruction of periodontal soft tissues as well as alveolar bone, and can be treated with anti‐inflammatory and bone‐protective agents to prevent disease progression. Caffeic acid phenethyl ester (CAPE) is a natural polyphenolic compound with anti‐inflammation, anti‐oxidation and bone tissue repair efficacy. In this work, we synthesized a thermosensitive hydrogel matrix of acetylated carboxymethyl chitosan (A‐CC), and firstly applied for periodontal local drug delivery. The biocompatible CAPE‐loaded A‐CC hydrogel (CAPE‐A‐CC) has the advantages of forming a drug depot in situ, sustained release and precisely improving the drug concentration in the lesion sites compared with traditional systemic administration. In addition, CAPE‐A‐CC could significantly inhibit the expression of inflammatory cytokines of TNF‐α, IL‐1β, IL‐6, and IL‐17 in macrophages, and increased the expression of alkaline phosphatase (ALP) in human periodontal ligament stem cells (hPDLSC) related to osteogenesis. This study develops a novel in situ thermosensitive hydrogel delivery system to improve the therapeutic potential of natural active ingredient for periodontitis therapy.

Intracranial In Situ Thermosensitive Hydrogel Delivery of Temozolomide Accomplished by PLGA-PEG-PLGA Triblock Copolymer Blending for GBM Treatment.

Glioblastoma (GBM) recurrence after surgical excision has grown to be a formidable obstacle to conquer. In this research, biodegradable thermosensitive triblock copolymer, poly(D, L–lactic acid–co–glycolic acid)–b–poly(ethylene glycol)–b–poly(D, L–lactic acid–co–glycolic acid (PLGA–PEG–PLGA) was utilized as the drug delivery system, loading with micronized temozolomide(micro-TMZ) to form an in situ drug–gel depot inside the resection cavity. The rheology studies revealed the viscoelastic profile of hydrogel under various conditions. To examine the molecular characteristics that affect gelation temperature, 1H–NMR, inverse gated decoupling 13C–NMR, and GPC were utilized. Cryo-SEM and XRD were intended to disclose the appearance of the hydrogel and the micro-TMZ existence state. We worked out how to blend polymers to modify the gelation point (Tgel) and fit the correlation between Tgel and other dependent variables using linear regression. To simulate hydrogel dissolution in cerebrospinal fluid, a membraneless dissolution approach was used. In vitro, micro-TMZ@PLGA–PEG–PLGA hydrogel exhibited Korsmeyer–Peppas and zero–order release kinetics in response to varying drug loading, and in vivo, it suppressed GBM recurrence at an astoundingly high rate. Micro-TMZ@PLGA–PEG–PLGA demonstrates a safer and more effective form of chemotherapy than intraperitoneal TMZ injection, resulting in a spectacular survival rate (40%, n = 10) that is much more than intraperitoneal TMZ injection (22%, n = 9). By proving the viability and efficacy of micro-TMZ@PLGA–PEG–PLGA hydrogel, our research established a novel chemotherapeutic strategy for treating GBM recurrence.

Long‐term delivery of etanercept mediated via a thermosensitive hydrogel for efficient inhibition of wear debris‐induced inflammatory osteolysis

AbstractPeriprosthetic wear debris‐induced aseptic loosening brings great pain to patients undergoing total joint arthroplasty. Tumor necrosis factor α (TNF‐α) is a pivotal cytokine involved in wear debris‐induced aseptic inflammation and subsequent osteolysis. Herein, we developed an injectable hydrogel system containing etanercept (ETN), a TNF‐α antagonist, to inhibit wear debris‐induced osteolysis. A set of thermosensitive poly(lactide‐co‐glycolide)‐b‐poly(ethylene glycol)‐b‐poly(lactide‐co‐glycolide) (PLGA–PEG–PLGA) copolymers with comparable molecular weights but different LA/GA molar ratios were synthesized by us, and their aqueous solutions presented sol‐to‐gel transitions along with the elevation of temperature. The LA/GA ratio not only impacted the phase transition temperature but also controlled the degradation rate of gel in vivo. The hydrogel with an appropriate in vivo degradation rate was employed to load and deliver ETN, and the drug introduction did not influence on its thermo‐induced gelation behavior. The loaded ETN was released from the gel depot in vitro in a sustained manner for up to 30 days. In a mouse bone‐implanted air pouch model, a single injection of the hydrogel formulation and subsequent sustained release of active ETN efficiently neutralized TNF‐α, resulting in significant suppression of titanium particles‐induced aseptic inflammation and subsequent osteolysis, and the effect was pronouncedly superior to that of an ETN solution.

Advanced delivery of leuprorelin acetate for the treatment of prostatic cancer

ABSTRACT Introduction Androgen-deprivation therapy (ADT) is the main therapy for patients with advanced and metastatic prostate cancer (PCa) and, in combination with radiotherapy, for patients with localized high-risk PCa. Due to its favorable tolerability among different treatments available for ADT, leuprorelin acetate is well established as the leading luteinizing hormone-releasing hormone (LHRH) analog. The development of second-generation leuprorelin acetate (LA) depot formulation (Eligard®, Recordati S.p.A) allowed a consistent and controlled release of leuprorelin between injections and a more efficient reduction of testosterone levels with respect to conventional LHRH agonists. Areas covered This work provides a summary of the biological and clinical rationale for using LA to manage PCa and presents the current evidence about the therapeutic activity of the LA gel depot formulation, used as an advanced leuprorelin acetate delivery method. Expert opinion Results of the registration studies and post-marketing clinical trials demonstrate that the LA gel depot provides long-term efficacy in the clinical practice and a good degree of tolerability. Overall, collected data suggest that the LA gel depot can represent the ADT reference therapy in advanced PCa.

A bio-responsive, cargo-catchable gel for postsurgical tumor treatment via ICD-based immunotherapy.

Tumor recurrence and metastasis after surgery remain challenges for tumor treatment. Strategy that can promote the immunogenicity, activate adaptative immune response and eliminate post-operative immunosuppression would be a promising way to achieve a desired clinical benefit. In this study, immunogenic cell death (ICD) priming anti-tumor adaptive immune response was executed to potentiate immune checkpoint blockade (ICB) therapy through the PD1/PDL1 pathway for postsurgical treatment. Here, we present a bio-responsive and cargo-catchable gel depot composed of pullulan and chitosan cross-linking through matrix metalloproteinase (MMP) sensitive peptide for co-delivery of anti-programmed death-ligand 1 antibody (aPDL1) and doxorubicin -encapsulated liposomes (DOX-Lip). This drug carrier showed expected ability to respond to the highly expressed MMP in postsurgical tumor microenvironment (TME). In vivo studies on 4T1 breast tumor mouse model demonstrated that the gel depot could efficiently prolong the mouse survival after tumor resection by preventing tumor recurrence and metastasis. The results suggested that ICD combining with PD1/PDL1 blockade based on the bio-responsive and cargo-catchable gel depot could facilitate the maturation of DCs and reverse the immunosuppressive environment in tumor resection area, thus amplifying the systemic anti-tumor immune response.

Injectable Hydrogel Containing Cowpea Mosaic Virus Nanoparticles Prevents Colon Cancer Growth.

Despite advances in laparoscopic surgery combined with neoadjuvant and adjuvant therapy, colon cancer management remains challenging in oncology. Recurrence of cancerous tissue locally or in distant organs (metastasis) is the major problem in colon cancer management. Vaccines and immunotherapies hold promise in preventing cancer recurrence through stimulation of the immune system. We and others have shown that nanoparticles from plant viruses, such as cowpea mosaic virus (CPMV) nanoparticles, are potent immune adjuvants for cancer vaccines and serve as immunostimulatory agents in the treatment or prevention of tumors. While being noninfectious toward mammals, CPMV activates the innate immune system through recognition by pattern recognition receptors (PRRs). While the particulate structure of CPMV is essential for prominent immune activation, the proteinaceous architecture makes CPMV subject to degradation in vivo; thus, CPMV immunotherapy requires repeated injections for optimal outcome. Frequent intraperitoneal (IP) injections however are not optimal from a clinical point of view and can worsen the patient's quality of life due to the hospitalization required for IP administration. To overcome the need for repeated IP injections, we loaded CPMV nanoparticles in injectable chitosan/glycerophosphate (GP) hydrogel formulations, characterized their slow-release potential, and assessed the antitumor preventative efficacy of CPMV-in-hydrogel single dose versus soluble CPMV (single and prime-boost administration). Using fluorescently labeled CPMV-in-hydrogel formulations, in vivo release data indicated that single IP injection of the hydrogel formulation yielded a gel depot that supplied intact CPMV over the study period of 3 weeks, while soluble CPMV lasted only for one week. IP administration of the CPMV-in-hydrogel formulation boosted with soluble CPMV for combined immediate and sustained immune activation significantly inhibited colon cancer growth after CT26 IP challenge in BALB/c mice. The observed antitumor efficacy suggests that CPMV can be formulated in a chitosan/GP hydrogel to achieve prolonged immunostimulatory effects as single-dose immunotherapy against colon cancer recurrence. The present findings illustrate the potential of injectable hydrogel technology to accommodate plant virus nanoparticles to boost the translational development of effective antitumor immunotherapies.

Novel Combined Lidocaine/Povidone Iodine Delivery System for Preintravitreal Injection.

Purpose: Intravitreal injection has become a popular treatment for various retina disorders and dramatically increased over the past few years. In traditional preintravitreal injection, the preparation steps are time consuming for practitioners who perform a significant number of injections per day. Besides, lidocaine gel (L-Gel) shows a potential absorption barrier on the antibacterial effect of povidone iodine (PI). Methods: In this study, we describe a L/PI gel system as an alternative approach to address these issues for traditional preinjection drug administration. Lidocaine and PI are loaded in a thermoresponsive gel instilled as a liquid to the lower fornix that transitions to a stable, solid gel depot. Results and Conclusion: The gel demonstrated decrease in conjunctival touch sensitivity and sufficient bacteria killing with a single step, suggesting a significant decrease in the time required and less potential for drug inhibition due to sequential administration.

This Month in Adult Urology

This Month in Adult Urology

Sustained Delivery of Lactoferrin Using Poloxamer Gels for Local Bone Regeneration in a Rat Calvarial Defect Model.

Lactoferrin (LF) is a multifunctional milk glycoprotein that promotes bone regeneration. Local delivery of LF at the bone defect site is a promising approach for enhancement of bone regeneration, but efficient systems for sustained local delivery are still largely missing. The aim of this study was to investigate the potential of the poloxamers for sustained delivery of LF to enhance local bone regeneration. The developed LF/poloxamer formulations were liquid at room temperature (20 °C) transforming to a sustained releasing gel depot at body temperature (37 °C). In vitro release studies demonstrated an initial burst release (~50%), followed by slower release of LF for up to 72 h. Poloxamer, with and without LF, increased osteoblast viability at 72 h (p < 0.05) compared to control, and the immune response from THP-1 cells was mild when compared to the suture material. In rat calvarial defects, the LF/poloxamer group had lower bone volume than the controls (p = 0.0435). No difference was observed in tissue mineral density and lower bone defect coverage scores (p = 0.0267) at 12 weeks after surgery. In conclusion, LF/poloxamer formulations support cell viability and do not induce an unfavourable immune response; however, LF delivery via the current formulation of LF200/poloxamer gel did not demonstrate enhanced bone regeneration and was not compatible with the rat calvarial defect model.

Thermosensitive Gels Used to Improve Microneedle-Assisted Transdermal Delivery of Naltrexone.

Transdermal delivery of naltrexone (NTX) can be enhanced using microneedles, although micropores generated this way can reseal by 48 h in humans, which prevents further drug delivery from a formulation. Poloxamer 407 (P407) is a thermosensitive polymer that may extend microneedle-assisted NTX delivery time by creating an in situ gel depot in the skin. We characterized gelation temperature, drug release, and permeation of P407 gels containing 7% NTX-HCl. To investigate microneedle effects on NTX-HCl permeation, porcine skin was treated with microneedles (600 or 750 μm length), creating 50 or 100 micropores. The formulations were removed from the skin at 48 h to simulate the effect of micropores resealing in vivo, when drug delivery is blunted. Gelation temperature increased slightly with addition of NTX-HCl. In vitro NTX-HCl release from P407 formulations demonstrated first order release kinetics. Microneedle treatment enhanced NTX-HCl permeation both from aqueous solution controls and P407 gels. Steady-state flux was overall lower in the P407 conditions compared to the aqueous solution, though ratios of AUCs before and after gel removal demonstrate that P407 gels provide more sustained release even after gel removal. This may be beneficial for reducing the required application frequency of microneedles for ongoing treatment.

An Overview: The Evaluation of Formation Mechanisms, Preparation Techniques and Chemical and Analytical Characterization Methods of the In Situ Forming Implants

One of the major developments of the last decade is the preparation of in situ implant formulations. Injectable, biocompatible and/or biodegradable polymer-based in situ implants are classified differently due to implant formation based on in vivo solid depot or formation mechanisms inducing liquid form, gel or solid depot. In this review, published studies to date regarding in situ forming implant systems were compiled and their formation mechanisms, materials and methods used, routes of administration, chemical and analytical characterizations, quality-control tests and in vitro dissolution tests were compared in Tables and were evaluated. There are several advantages and disadvantages of these dosage forms due to the formation mechanism, polymer and solvent type and the ratio used in formulations and all of these parameters have been discussed separately. In addition, new generation systems developed to overcome the difficulties encountered in in situ implants have been evaluated. There are some approved products of in situ implant preparations that can be used for different indications available on the market and the clinical phase studies nowadays. In vitro and in vivo data obtained by the analysis of the application of new technologies in many studies evaluated in this review showed that the number of approved drugs to be used for various indications would increase in the future.

Controlled release of ciprofloxacin and ceftriaxone from a single ototopical administration of antibiotic-loaded polymer microspheres and thermoresponsive gel.

Acute otitis media (AOM) is the main indication for pediatric antibiotic prescriptions, accounting for 25% of prescriptions. While the use of topical drops can minimize the administered dose of antibiotic and adverse systemic effects compared to oral antibiotics, their use has limitations, partially due to low patient compliance, high dosing frequency, and difficulty of administration. Lack of proper treatment can lead to development of chronic OM, which may require invasive interventions. Previous studies have shown that gel-based drug delivery to the ear is possible with intratympanic injection or chemical permeation enhancers (CPEs). However, many patients are reluctant to accept invasive treatments and CPEs have demonstrated toxicity to the tympanic membrane (TM). We developed a novel method of delivering therapeutics to the TM and middle ear using a topical, thermoresponsive gel depot containing antibiotic-loaded poly(lactic-co-glycolic acid) microspheres. Our in vitro and ex vivo results suggest that the sustained presentation can safely allow therapeutically relevant drug concentrations to penetrate the TM to the middle ear for up to 14 days. Animal results indicate sufficient antibiotic released for treatment from topical administration 24h after bacterial inoculation. However, animals treated 72h after inoculation, a more clinically relevant treatment practice, displayed spontaneous clearance of infection as is also often observed in the clinic. Despite this variability in the disease model, data suggest the system can safely treat bacterial infection, with future studies necessary to optimize microsphere formulations for scaled up dosage of antibiotic as well as further investigation of the influence of spontaneous bacterial clearance and of biofilm formation on effectiveness of treatment. To our knowledge, this study represents the first truly topical drug delivery system to the middle ear without the use of CPEs.

Meticulous Doxorubicin Release from pH-Responsive Nanoparticles Entrapped within an Injectable Thermoresponsive Depot.

The dual stimuli-controlled release of doxorubicin from gel-embedded nanoparticles is reported. Non-cytotoxic polymer nanoparticles are formed from poly(ethylene glycol)-b-poly(benzyl glutamate) that, uniquely, contain a central ester link. This connection renders the nanoparticles pH-responsive, enabling extensive doxorubicin release in acidic solutions (pH 6.5), but not in solutions of physiological pH (pH 7.4). Doxorubicin-loaded nanoparticles were found to be stable for at least 31 days and lethal against the three breast cancer cell lines tested. Furthermore, doxorubicin-loaded nanoparticles could be incorporated within a thermoresponsive poly(2-hydroxypropyl methacrylate) gel depot, which forms immediately upon injection of poly(2-hydroxypropyl methacrylate) in dimethyl sulfoxide solution into aqueous solution. The combination of the poly(2-hydroxypropyl methacrylate) gel and poly(ethylene glycol)-b-poly(benzyl glutamate) nanoparticles yields an injectable doxorubicin delivery system that facilities near-complete drug release when maintained at elevated temperatures (37 °C) in acidic solution (pH 6.5). In contrast, negligible payload release occurs when the material is stored at room temperature in non-acidic solution (pH 7.4). The system has great potential as a vehicle for the prolonged, site-specific release of chemotherapeutics.

Mild\xa0photothermal therapy potentiates anti-PD-L1 treatment for immunologically cold tumors via an all-in-one and all-in-control strategy

One of the main challenges for immune checkpoint blockade antibodies lies in malignancies with limited T-cell responses or immunologically “cold” tumors. Inspired by the capability of fever-like heat in inducing an immune-favorable tumor microenvironment, mild photothermal therapy (PTT) is proposed to sensitize tumors to immune checkpoint inhibition and turn “cold” tumors “hot.” Here we present a combined all-in-one and all-in-control strategy to realize a local symbiotic mild photothermal-assisted immunotherapy (SMPAI). We load both a near-infrared (NIR) photothermal agent IR820 and a programmed death-ligand 1 antibody (aPD-L1) into a lipid gel depot with a favorable property of thermally reversible gel-to-sol phase transition. Manually controlled NIR irradiation regulates the release of aPD-L1 and, more importantly, increases the recruitment of tumor-infiltrating lymphocytes and boosts T-cell activity against tumors. In vivo antitumor studies on 4T1 and B16F10 models demonstrate that SMPAI is an effective and promising strategy for treating “cold” tumors.

ROS‐Sensitive Degradable PEG–PCL–PEG Micellar Thermogel

A reactive oxygen species (ROS)-sensitive degradable polymer would be a promising material in designing a disease-responsive system or accelerating degradation of polymers with slow hydrolysis kinetics. Here, a thermogelling poly(ethylene glycol)-polycaprolactone-poly(ethylene glycol) (PEG-PCL-PEG or EG12 -CL20 -EG12 ) triblock copolymer with an oxalate group at the middle of the polymer is reported. The polymers form micelles with an average size of 100 nm in water. Thermogelation is observed in a concentration range of 8.0-37.0 wt%. In particular, the aqueous PEG-PCL-PEG triblock copolymer solutions are in a gel state at 37 °C in a concentration range of 25.0-37.0 wt%, whereas the aqueous PEG-PCL diblock copolymer solutions are in a sol state in the same concentration range at 37 °C. Thus, the gel depot could dissolve out once degradation of the triblock copolymers occurs at the oxalate group as confirmed by the in vitro experiment. In vivo gel formation is confirmed by injecting an aqueous PEG-PCL-PEG solution (36.0 wt%) into the subcutaneous layer of rats. The gel completely disappears in 21 d. A model polypeptide drug (cyclosporine A) is released over 21 d from the in situ formed gel. The micelle-based thermogel of PEG-PCL-PEG with ROS-triggering degradability is a promising injectable material for biomedical applications.

A Dual-Bioresponsive Drug-Delivery Depot for Combination of Epigenetic Modulation and Immune Checkpoint Blockade.

Patients with advanced melanoma that is of low tumor-associated antigen (TAA) expression often respond poorly to PD-1/PD-L1 blockade therapy. Epigenetic modulators, such as hypomethylation agents (HMAs), can enhance the antitumor immune response by inducing TAA expression. Here, a dual bioresponsive gel depot that can respond to the acidic pH and reactive oxygen species (ROS) within the tumor microenvironment (TME) for codelivery of anti-PD1 antibody (aPD1) and Zebularine (Zeb), an HMA, is engineered. aPD1 is first loaded into pH-sensitive calcium carbonate nanoparticles (CaCO3 NPs), which are then encapsulated in the ROS-responsive hydrogel together with Zeb (Zeb-aPD1-NPs-Gel). It is demonstrated that this combination therapy increases the immunogenicity of cancer cells, and also plays roles in reversing immunosuppressive TME, which contributes to inhibiting the tumor growth and prolonging the survival time of B16F10-melanoma-bearing mice.

Enhanced local cancer therapy using a CA4P and CDDP co-loaded polypeptide gel depot.

Cancer combination therapy based on drug co-delivery systems provides an effective strategy for enhancing treatment efficacy and reducing side effects. In this work, a new strategy through co-delivery of combretastatin A4 disodium phosphate (CA4P) and cisplatin (CDDP) was developed for the local treatment of colon cancer, through an in situ thermo-gelling hydrogel (mPEG-b-PELG). The results indicated that this material possessed concentration-dependent thermogelling properties and tunable in vivo biodegradability. Also, the drug loaded gel could regulate the in vitro drug release behaviors of both CDDP and CA4P, which promoted the in vivo vessel disrupting effects of CA4P compared with a free drug after local treatment for 48 h. Although the drug co-loaded gel induced less in vitro cell death compared with the free drug co-treated group, this drug co-loaded gel depot showed the highest antitumor efficacy compared with the other experimental groups after peritumoral injection toward C26 tumor bearing mice.

Injectable Bioresponsive Gel Depot for Enhanced Immune Checkpoint Blockade.

Although cancer immunotherapy based on immune checkpoint inhibitors holds great promise toward many types of cancers, several challenges still remain, associated with low objective response of patient rate as well as systemic side effects. Here, a combination immunotherapy strategy is developed based on a thermogelling reactive oxygen species (ROS)-responsive polypeptide gel for sustained release of anti-programmed cell death-ligand 1 antibody and dextro-1-methyl tryptophan, inhibitor of indoleamine-2,3-dioxygenase with leveraging the ROS level in the tumor microenvironment. This bioresponsive gel depot can effectively reduce the local ROS level and facilitate release of immunotherapeutics, which leads to enhanced anti-melanoma efficacy in vivo.