In preclinical studies, we investigated a novel mechanism of in situ vaccination in lymphoma. Radiation therapy (RT) can induce abscopal responses in lymphoma models, but this has not translated into clinical efficacy. We hypothesized that immune stimulation with cytosine guanine dinucleotide (CpG) deoxynucleotides could enhance abscopal effects induced by RT or photothermal therapy (PTT), which has been shown to have an immune stimulatory effect in solid tumors but has not been studied in lymphoma. We designed a branched gold nanoparticle (NP) platform to carry CpG deoxynucleotides while maintaining PTT function and compared the immunologic profile of the tumor microenvironment after PTT or RT in a dual-flank lymphoma model. One flank was treated with CpG deoxynucleotides with RT or PTT, and the other tumor was left untreated. We found that the CpG deoxynucleotide/PTT group had significant reduction in growth in both treated (primary) and untreated (secondary) tumors, suggesting an improved abscopal response, with a concomitant increase in CD8/CD4 and cytotoxic T-cell/regulatory T-cell ratios in both primary and secondary tumors compared with CpG deoxynucleotides/RT. Dendritic cells in primary and secondary draining lymph nodes had increased maturation markers in the CpG deoxynucleotide/PTT group, and the effector memory T cells (both CD4 and CD8) in the secondary tumor and spleen were increased, suggesting a systemic vaccination effect. These data suggest that in a lymphoma model, PTT using a CpG deoxynucleotide NP platform resulted in enhanced in situ vaccination and abscopal response compared with RT.

Hyaluronic acid (HA) is being actively studied as a drug carrier due to its favorable properties and functional groups for chemical modification. Despite its high functionality, p K a of HA (3–4, carboxyl groups) is inappropriate to release the drugs at the tumor pH. For the development of a pH-sensitive nanocarrier based on HA for anticancer drugs, HA- graft -imidazole-dodecylamine (HID) was synthesized. The HID formed a stable nanocarrier with ca. 151 nm size and low critical association concentration (CAC) at pH 7.4 and was destabilized in acidic conditions (pH 6.8–6.0) with increased size and CAC. Doxorubicin loaded HID nanocarriers (DHNs) exhibited pH-dependent drug release with the structural change by the deprotonation and protonation of the imidazole groups in HID. The low CAC of HID at physiological pH and the pH-dependent drug release would confer high stability and prevent drug loss during systemic circulation. It might reduce the toxicity to normal tissue at physiological pH. In addition, tumor extracellular pH and early endosomal pH environments triggered the disintegration of nanocarriers, switching the drug release higher than in other normal tissues and blood. Consequently, HID could be a biocompatible pH-sensitive drug carrier for cancer chemotherapy.

Lutein has been used as a dietary supplement for the treatment of eye diseases, especially age-related macular degeneration. For oral formulations, we investigated lutein stability in artificial set-ups mimicking different physiological conditions and found that lutein was degraded over time under acidic conditions. To enhance the stability of lutein upon oral intake, we developed enteric-coated lutein solid dispersions (SD) by applying a polymer, hydroxypropyl methylcellulose acetate succinate (HPMCAS-LF), through a solvent-controlled precipitation method. The SD were characterized in crystallinity, morphology, and drug entrapment. In the dissolution profile of lutein SD, a F80 formulation showed resistance toward the acidic environment under simulated gastric conditions while exhibiting a bursting drug release under simulated intestinal conditions. Our results highlight the potential use of HPMCAS-LF as an effective matrix to enhance lutein bioavailability during oral delivery and to provide novel insights into the eye-care supplement industry, with direct benefits for the health of patients.

Natural killer (NK) cell-based immunotherapy has been considered a promising cell-based cancer treatment strategy with low side effects for early tumors and metastasis. However, the therapeutic efficacy is generally low in established solid tumors. Ex vivo activation of NK cells with exogenous cytokines is often essential but ineffective to generate high doses of functional NK cells for cancer treatment. Image-guided local delivery of NK cells is also suggested for the therapy. However, there is a lack of noninvasive tools for monitoring NK cells. Herein, magnetic nanocomplexes are fabricated with clinically available materials (hyaluronic acid, protamine, and ferumoxytol; HAPF) for labeling NK cells. The prepared HAPF-nanocomplexes effectively attach to the NK cells (HAPF-NK). An exogenous magnetic field application effectively achieves magneto-activation of NK cells, promoting the generation and secretion of lytic granules of NK cells. The magneto-activated HAPF-NK cells also allow an MR image-guided NK cell therapy to treat hepatocellular carcinoma (HCC) solid tumors via transcatheter intra-arterial infusion. Suppressed tumor growth after the treatment of IA infused magneto-activated NK cells demonstrated a potential enhanced therapeutic efficacy of image guided local delivery of magneto-activated HAPF-NK cells. Given the potential challenges of NK cell cancer immunotherapy against established solid tumors, the effective NK cell labeling with HAPF, magneto-activation, and MRI contrast effect of NK cells will be beneficial to enhance the NK cell-therapeutic efficacy in various cancers.

Introduction: Although radiation therapy (RT) has limited clinical efficacy for advanced stage lymphoma, RT has been used as a method for in situ vaccination to generate an abscopal effect. Frank et al. combined low dose RT with class C CpG deoxynucleotides (CpGs) for the treatment of advanced stage indolent lymphoma (Cancer Discovery, 2018). CpGs are toll-like receptor 9 agonists and activates the innate immune system. The overall response rate was high (89.6%) in this trial but only 7 of 29 patients (pts) (24%) had partial response and 1 had a complete response (3%). Unlike external beam RT, photothermal therapy (PTT) using gold nanoparticles (AuNPs) can generate a strong in situ vaccination effect by heat ablation of tumors (Bear et al. PLOS One, 2013). (Figure 1A) The ablation induced increased CD8 T cell infiltration in non-treated tumors and dendritic cell maturation in the draining lymph nodes, but it also increased systemic immune suppression via increased myeloid suppressor cells (MDSCs) in a melanoma model. MDSCs differentiate into active macrophages when stimulated by CpGs. Other PTT methods have been evaluated in combination with check-point inhibitors with success in solid tumor murine models (Liu et al. Immunotherapy 2018; Chen et al. Nature Comm. 2016). Therefore, we hypothesized that PTT in combination with CpG can elicit a stronger the in situ vaccination effect compared with RT with CpG in a murine lymphoma model. Methods: PTT is achieved by exciting branched AuNP (BNP) with a near infrared (NIR) laser (808nm). For the dual lymphoma model, A20 lymphoma cells in Matrigel were injected into the right flank of BALB/c mice to establish a primary tumor. Three days later, a secondary tumor was established in the left flank. When the tumor reached 5 mm in diameter, PTT with CpG (sequence 2395) or RT (10Gy) with CpG of the primary tumor were performed. Control groups included PTT without CpG, CpG only, BNP with CpG without PTT, and PBS. Sixteen days after PTT or RT, mice are euthanized and flow cytometry was performed on the primary tumor, secondary tumor, spleen, and draining lymph nodes and analyzed for T cells and dendritic cells (DC). Results: Within 5 minutes after PTT, the temperature of the treated primary tumor reached almost 60°C. The primary tumor growth was inhibited for both RT/CpG and PTT/CpG treatment groups compared with the PBS control group. RT/CpGs treated primary tumors demonstrated re-growth 14 days (D14) after treatment while PTT/CpG treated tumors did not (p<0.01). For the non-treated secondary tumors, the RT/CpG group had delayed but continued tumor growth compared with the PBS control group, while PTT/CpG maintained tumor growth suppression, with the growth curves separating from the RT/CpG group as early as D9 (p<0.01). (Figure 1B) At the end of the study (D16), PTT/CpG group secondary tumors (344mm3) were almost a third of the size of RT/CpG group (958 mm3) (p=0.006). To further investigate the mechanism for the improved abscopal effect, we compared the differences in immune response between RT/CpG and PTT/CpG treatment groups. The amount of mature DCs (CD80+CD86+ in CD11c+ cells) in draining lymph nodes of both the PTT/CpG treated tumor (14.9%) and non-treated tumor (15.9%) were significantly higher than the RT/CpG group (8.78%, p=0.006; 10.2%, p=0.007, respectively). For the non-treated tumor, the PTT/CpG group had significantly increased amounts of CD8 (62.6%) T cell infiltration compared with the RT/CpG group (45.4%, p=0.022) as well as a higher CD8 to CD4 T cell ratio (1.91) compared with the RT/CpG group (0.95, p=0.042). Effector memory T cells in the secondary tumor (44.6%) and spleen (29.9%) for the PTT/CpG group was also higher than the RT/CpG group (23.2%, p=0.008; 21.2%, p=0.011, respectively). These changes suggest a significant exaggeration in T-cell education, which consequently results in suppression of lymphoma tumor growth. Conclusion: Overall, we demonstrated as a proof-of-concept that PTT/CpG generated a stronger in situ vaccination effect when compared with RT/CpG therapy in lymphoma. In addition, we have described the immune responses after PTT in combination with CpG in a lymphoma model, showing increased dendritic cell maturation and T cell mediated immune response. Further studies including dosing schedule, combination therapy with check-point inhibitors or other immune based treatments will move this technology closer to clinical practice. Disclosures Gordon: Zylem Biosciences: Patents & Royalties: Patents, No Royalties.

The development of an oral formulation that ensures increased bioavailability of drugs is a great challenge for pharmaceutical scientists. Among many oral formulation systems, a drug delivery system employing superporous networks was developed to provide a prolonged gastro-retention time as well as improved bioavailability of drugs with a narrow absorption window in the gastrointestinal tract. Superporous networks (SPNs) were prepared from chitosan by crosslinking with glyoxal and poly(vinyl alcohol) (PVA). The SPNs showed less porosity and decreased water uptake with an increase in the crosslinking density and content of PVA. Gastro-retentive tablets (GRTs) were formulated using hydroxypropyl methylcellulose (HPMC, a hydrophilic polymer) and the prepared SPNs. Ascorbic acid (AA), which is mainly absorbed in the proximal part of the small intestine, was selected as a model drug. The formulated GRTs exhibited no floating lag time and stayed afloat until the end of the dissolution test. The in vitro drug release from the GRTs decreased with a decrease in the water uptake of the SPNs. The profile of drug release from the GRTs corresponded to the first-order and Higuchi drug-release models. Overall, floating tablets composed of the SPNs and HPMC have potential as a favorable platform to ensure sustained release and improved bioavailability of drugs that are absorbed in the proximal part of the small intestine.

Combination therapy is considered to be a promising strategy for improving the therapeutic efficiency of cancer treatment. In this study, an on-demand pH-sensitive nanocluster (NC) system was prepared by the encapsulation of gold nanorods (AuNR) and doxorubicin (DOX) by a pH-sensitive polymer, poly(aspartic acid-graft-imidazole)-PEG, to enhance the therapeutic effect of chemotherapy and photothermal therapy. At pH 6.5, the NC systems formed aggregated structures and released higher drug amounts while sustaining a stable nano-assembly, structured with less systemic toxicity at pH 7.4. The NC could also increase antitumor efficacy as a result of improved accumulation and release of DOX from the NC system at pHex and pHen with locally applied near-infrared light. Therefore, an NC system would be a potent strategy for on-demand combination treatment to target tumors with less systemic toxicity and an improved therapeutic effect.

Nanocarriers with pH-sensitive functionality are of great interest in the development of pH-dependent drug release compounds in acidic tumor microenvironments. A new polyelectrolyte block copolymer, poly[(benzyl-L-aspartate)-co-(N-(3-aminopropyl) imidazole-L-aspartamide)]-poly(ethylene glycol) (PABI-PEG), was prepared by one-step modulation to produce pH-sensitive nanocarriers. PABI-PEG formed a stable nanocarrier at pH values above 7.4 and was destabilized in acidic conditions (pH 6.5) through the protonation of the imidazole groups. Docetaxel loaded micelle (DLM) exhibited pH-dependent drug release through structural conversion due to the protonation of the imidazole groups on the PABI block. The critically low micelle concentration of PABI-PEG at physiological pH and the pH-dependent drug release would result to high stability and restrict drug loss during systemic circulation which may lower the toxicity of normal tissue to physiological pH. Additionally, the extracellular tumor pH (<7.0) and early endosomal pH (<6.5) environments triggered the disintegration of micelles, producing higher drug release compared to other normal tissues and blood (pH 7.4). Therefore, PABI-PEG may be a pHsensitive drug delivery method for cancer chemotherapy.

Currently, one of the most important challenges in the development of nanotechnology-based anticancer treatments is the failure of nanoparticles to escape from the endo-lysosomal compartment.

Background: A very common and simple method (known as the blending method) to formulate drug delivery systems with required properties is to physically mix amphiphilic block copolymers with different hydrophobicity. In addition to its simplicity, this blending strategy could help avoid the time and effort involved in the synthesis of block copolymers with the desired structure required for specific drug formulations. Purpose: We used the blending strategy to design a system that could overcome the problem of high hydrophobicity and be a good candidate for drug product development using PEG-PLA-PEG triblock copolymers. Methods: Two types of PEG-PLA-PEG triblock copolymers with similar (long) PLA molecular weights (MWs) and different PEG MWs were synthesized. The micellar formulations were prepared by blending the two block copolymers in various ratios. The size and stability of the blending systems were subsequently investigated to optimize the formulations for further studies. The loading properties of doxorubicin or paclitaxel into the optimized blending system were compared to that in mono systems (systems composed of only a single type of triblock copolymer). In vitro and in vivo anti-cancer effects of the preparations were evaluated to assess the use of the blending system as an optimal nanomedicine platform for insoluble anticancer agents. Results: The blending system (B20 system) with an optimized ratio of the triblock copolymers overcame the drawbacks of mono systems. Drug uptake from the drug-loaded B20 system and its anticancer effects against KB cells were superior compared to those of free drugs (doxorubicin hydrochloride and free paclitaxel). In particular, doxorubicin-loaded B20 resulted in extensive doxorubicin accumulation in tumor tissues and significantly higher in vivo anti-cancer effects compared to free doxorubicin. Conclusion: The blending system reported here could be a potential nanoplatform for drug delivery due to its simplicity and efficiency for pharmaceutical application.

BACKGROUND:Blending micellar systems of different types of polymers has been proposed as an efficient approach for tailor-made drug formulations. The lamellar structure of hydrophobic polymers may provide a high drug loading capacity, and hydrophilic polymers may provide good colloidal stability.METHODS:In this study, the anticancer model drug docetaxel was loaded onto a nanosized blending micellar system with two pluronics (L121/F127). To achieve increased antitumor activity, the cyclic arginine-glycine-aspartic acid tripeptide (cRGD) as an active tumor targeting ligand was conjugated to the blending system.RESULTS:The docetaxel-loaded Pluronic blending system exhibited a higher drug loading capacity than that of F127 and showed high colloidal stability with a spherical structure. cRGD conjugates demonstrated enhanced drug cellular uptake and anticancer activity against αvβ3 integrin-overexpressing U87MG cancer cells. In vivo animal imaging also revealed that the prepared cRGD-conjugated nanoparticles effectively accumulated at the targeted tumor site through an active and passive targeting strategy.CONCLUSION:Accordingly, the prepared nanosized system shows potential as a tailor-made, active targeting, nanomedicinal platform for anticancer therapy. We believe that this novel nanoplatform will provide insights for advancement of tumor therapy.

Docetaxel (DTX)-loaded polymeric micelles (DTBM) were formulated using the triblock copolymer, poly(ethylene glycol)-polylactide-poly(ethylene glycol) (PEG-PLA-PEG), to comprehensively study their pharmaceutical application as anticancer nanomedicine. DTBM showed a stable formulation of anticancer nanomedicine that could be reconstituted after lyophilization (DTBM-R) in the presence of PEG 2000 and D-mannitol (Man) as surfactant and protectant, respectively. DTBM-R showed a particle size less than 150nm and greater than 90% of DTX recovery after reconstitution. The robustly formed micelles might minimize systemic toxicity due to their sustained drug release and also maximize antitumor efficacy through increased accumulation and release of DTX from the micelles. From the pharmaceutical development point of view, DTBM-R showing successful reconstitution could be considered as a potent nanomedicine for tumor treatment.

ABSTRACTpH‐sensitive nanogels (NGs) based on poly(aspartic acid‐graft‐imidazole)‐poly(ethylene glycol) were developed using linear PEG with different molecular weights (2000 and 4000 Da) as crosslinkers. The pH‐sensitive NGs showed reversible size changes during continuously alternating pH changes. The anticancer treatment potential of pH‐sensitive NGs was studied using a model drug, irinotecan (IRI). IRI‐loaded NGs (ILNs) showed different drug release kinetics in acidic versus neutral pH, in addition to pH‐dependent cytotoxicity. Due to its longer crosslinker, ILN 4 (crosslinked with PEG 4000) showed faster IRI release and a greater magnitude of IRI release than ILN 2 (crosslinked with PEG 2000), resulting in greater cytotoxicity against HCT 116 colorectal cancer cells. These pH‐sensitive NGs could potentially be used in cancer treatment by mediating the accumulation and release of IRI from ILNs in the acidic tumor environment and by reducing systemic toxicity due to reversible swelling–shrinkage. © 2018 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2018, 135, 46268.

Photo-Chemo combination therapy has been intensively investigated for treatment of cancers, especially multidrug resistance cancer. However, antagonistic interactions between chemo-drugs and photosensitizers are frequently reported, and drugs doses and treatment sequences have been changed to overcome the problems. We observed the antagonistic effect by a decrease in singlet oxygen generation from the photosensitizer when Dox was in close physical proximity. To control the distance between Dox and the photosensitizer, we developed a novel pH-sensitive poly ionomer complex system composed of PEG-PLL(-g-Ce6) [Chlorin e6 grafted poly(ethylene glycol)-poly(l-lysine)] and PEG-PLL(-g-DMA)-PLA [2,3-dimethylmaleic anhydride grafted poly(ethylene glycol)-poly(l-lysine)-poly(lactic acid)] and evaluated this system with regard to singlet oxygen generation and antiproliferative activity against MCF-7/Dox cells. Enhanced singlet oxygen generation and antiproliferative activities were observed in vitro and in vivo for the poly ionomer complex system compared to PEG-PLL(-g-Ce6)-PLA/Dox due to the change in distance between Dox and Ce6 in the PIC system under acidic conditions. Our results highlight the importance of interactions between co-loaded drugs in combination therapy, and provide new insights into design principles for tailor-made nanomedicine platforms.

Triblock copolymers have been widely used as a material for developing drug delivery systems (DDS). In terms of architecture, triblock copolymers could be classified into symmetric and asymmetric triblock copolymers. Different types of nano-sized structures such as star micelles, flower-like micelles, and polymer vesicles could be prepared from these block copolymers, which would be very potential in delivering various types of agents such as chemical drugs, genes, and contrast agents. Additionally, the nano-sized carriers have been fabricated for environmentally sensitive (pH-sensitive or temperature sensitive) DDS or for enhancing the gene delivery efficiency. Due to their versatility in structures and drug delivery capacity, the application of triblock copolymers would definitely be expanded in near future.

Interferon-α (IFN-α) has been widely used for the treatment of infections due to the hepatitis C virus (HCV). Because of the short half-life of IFN-α in serum, it must be administered three times per week. To increase the half-life of IFN-α, the immunoglobulin G4 (IgG4) Fc fragment (HMC001) was conjugated with human IFN-α-2b to develop a long-acting IFN-α-2b, HM10660A. An analysis of the antiviral efficacy of HM10660A in a human hepatocyte-engrafted mouse model found that HM10660A reduced serum HCV titers more effectively than a commercially available peginterferon α-2a (PEGASYS®) and IFN-α-2b. Pharmacokinetic (PK) and pharmacodynamic (PD) studies of HM10660A using monkeys demonstrated that the half-life of HM10660A was approximately 2-fold longer than commercially available peginterferon α-2a, which is approved for a once-weekly regimen. Moreover, the IFN-mediated induction profiles of neopterin and 2′, 5′-oligoadenylate synthase (OAS) in normal cynomolgus monkeys indicated that HM10660A had enhanced antiviral activity and a prolonged duration of action compared with peginterferon α-2a. Considering the improved PK and PD properties, HM10660A can most likely be dosed every two or four weeks, providing superior antiviral efficacy and convenience for patients with HCV.

OBJECTIVES:Solid dispersion formulations have attracted attention to improve solubility and bioavailability of water-insoluble drugs. In this study, the variation of solubility and bioavailability by different preparation methods were studied using itraconazole (ITZ) solid dispersions.METHODS:Itraconazole solid dispersions were prepared by a solvent-controlled precipitation method (SCPM) using HPMCAS-LF, HCl antisolvent or a spray-drying method (SDM) for comparison. Dissolution tests by pH transition and pharmacokinetic study using male Sprague Dawley rats were conducted.KEY FINDINGS:Itraconazole solid dispersion dissolution tests by pH transition exhibited better dissolution compared to naive ITZ, limited dissolution in acidic conditions and a burst release at neutral pH. The ITZ solid dispersions by SCPM indicated a smaller-sized particle dispersion, limited dissolution at acidic pH and a higher release at neutral pH compared to those by SDM, suggesting that the increased protonation of anionic polymers and HPMCAS-LF by acidic antisolvent could form a tighter hydrophobic aggregation with ITZ in solid dispersions. ITZ solid dispersion prepared by SCPM also showed improved ITZ absorption in male Sprague Dawley rats compared to SDM and naïve ITZ.CONCLUSIONS:This study suggests that the SCPM method can be widely used for solid dispersion preparations due to improved dissolution and PK profile.

Polyelectrolyte has been proposed as an efficient approach for various types of drug formulations. However, one drawback of using the conventional polyelectrolyte for drug delivery is its dissociation in in vivo conditions by counter ions due to the lack of self-assembling aggregation force. In this study, we reported a stable nanoplatform based on triblock co-polyelectrolyte composed of a poly(ethylene glycol), poly(l-lysine), and poly(lactic acid). These co-polyelectrolytes formed stable aggregates through the hydrophobic interaction of PLA and showed consistent particle sizes under a high salt concentration. In addition, the doxorubicin (Dox) loaded triblock co-polyelectrolyte demonstrated enhanced cellular uptake and drug cytotoxicity with a positive charge from the poly(l-lysine) layer. In vivo, the triblock aggregates exhibited intensive accumulation at the targeted tumor site for 24 h with good antitumor therapeutic efficacy. Therefore, the prepared stable triblock co-polyelectrolyte may have considerable potential as a nanomedicinal platform for anticancer and multi-drug combination therapy.

pH sensitive nanocarriers have showed the tumor targeted drug delivery and release by enhanced permeability and retention effect and responsiveness of acidic pH conditions of the solid tumors. The protonation and deprotonation of the nanocarriers including pH sensitive polyelectrolytes has been considered as a major mechanism, resulting in substantial conformational change in response to marginal change in the tumor pH conditions. Considering the biocompatible properties for pharmaceutical applications, poly(amino acid) showed low polymer toxicity and the formation of versatile systems for advanced antitumor therapeutic effects. In here, recent researches using cationic (histidine, lysine, and arginine), anionic (aspartic acid and glutamic acid), and zwitterionic amino acid were summarized. Furthermore, pH sensitive nanogels with unique functionality and other pH sensitive polymers were briefly described. pH sensitive nanogels showed reversible drug release by pH cyclization minimizing drug loss and enhancing drug release. In the future, non-spherical pH sensitive nanocarriers would be anticipated to improve the therapeutic effect by long circulation and stealth effect by low phagocytosis.