Sustained Release Formulation of Hydroxypropyl-ß-cyclodextrin Eye Drops Using Xanthan Gum.

Bietti's crystalline dystrophy (BCD) is an autosomal recessive chorioretinal degeneration caused by mutations in the CYP4V2 gene. It is characterized by cholesterol accumulation and crystal-like deposits in the retinas. Hydroxypropyl-ß-cyclodextrin (HP-ß-CyD) exerts therapeutic effects against BCD by reducing lysosomal dysfunction and inhibiting cytotoxicity in induced pluripotent stem cell (iPSC)-RPE cells established from patient-derived iPS cells. However, the ocular retention of HP-ß-CyD is low and needs to be improved. Therefore, this study used a viscous agent to develop a sustained-release ophthalmic formulation containing HP-ß-CyD. Our results suggest that HP-ß-CyD-containing xanthan gum has a considerably higher sustained release capacity than other viscous agents, such as methylcellulose and sodium alginate. In addition, the HP-ß-CyD-containing xanthan gum exhibited pseudoplastic behavior. It was less cytotoxic to human retinal pigment epithelial cells compared with HP-ß-CyD alone. Furthermore, the slow release of HP-ß-CyD from xanthan gum caused a sustained decrease in free intracellular cholesterol. These results suggest that xanthan gum is a useful substrate for the sustained release formulation of HP-ß-CyD, and that HP-ß-CyD-containing xanthan gum has potential as an eye drop for BCD treatment.

Colletofragarone A2 Inhibits Cancer Cell Growth In Vivo and Leads to the Degradation and Aggregation of Mutant p53.

Mutant p53 not only loses its original tumor suppressor function but also acquires new abilities regarding oncogenic progression. Therefore, the strategy of targeting mutant p53 has attracted attention for cancer therapy. We isolated colletofragarone A2 (CF) from the fungus Colletotrichum sp. (13S020), which decreases mutant p53 levels in cells, and herein examine its effect on mutant p53. CF showed more potent cytotoxic activities on cells with p53R175H structural mutants than those with different p53 statuses such as a DNA-contact mutant, wild-type, and null cells. CF markedly decreased tumor cell growth in vivo using a mouse xenograft model with HuCCT1 (p53R175H) cells. Cotreatment of SK-BR-3 (p53R175H) cells with CF and cycloheximide decreased mutant p53 levels by promoting p53 degradation. In the presence of MG-132, CF induced the accumulation of the aggregated mutant p53. These results suggest that CF inhibits the function of molecular chaperones such as HSP90.

Feasibility Study of Dendrimer-Based TTR-CRISPR pDNA Polyplex for Ocular Amyloidosis in Vitro.

Hereditary amyloidgenic transthyretin (ATTR) amyloidosis is caused by a genetic point-mutated transthyretin such as TTR Val30Met (TTR V30M), since it forms protein aggregates called amyloid resulting in the tissue accumulation and functional disorders. In particular, ATTR produced by retinal pigment epithelial cells often causes ATTR ocular amyloidosis, which elicits deterioration of ocular function and ultimately blindness. Therefore, development of novel therapeutic agents is urgently needed. Genome-editing technology using Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated proteins (CRISPR-Cas9) system is expected to be a therapeutic approach to treat genetic diseases, such as ATTR amyloidosis caused by a point mutation in TTR gene. Previously, we reported that glucuronylglucosyl-ß-cyclodextrin conjugated with a polyamidoamine dendrimer (CDE) had excellent gene transfer ability and that underlying dendrimer inhibited TTR aggregation. Conversely, folate receptors are known to be highly expressed in retina; thus, folate has potential as a retinal target ligand. In this study, we prepared a novel folate-modified CDE (FP-CDE) and investigated its potential as a carrier for the retinal delivery of TTR-CRISPR plasmid DNA (pDNA). The results suggested that FP-CDE/TTR-CRISPR pDNA could be taken up by retinal pigment epithelial cells via folate receptors, exhibited TTR V30M amyloid inhibitory effect, and suppressed TTR production via the genome editing effect (knockout of TTR gene). Thus, FP-CDE may be useful as a novel therapeutic TTR-CRISPR pDNA carrier in the treatment of ATTR ocular amyloidosis.

Hepatocyte-Targeted Delivery of siRNA Polyplex with PEG-Modified Lactosylated Dendrimer/Cyclodextrin Conjugates for Transthyretin-Related Amyloidosis Therapy.

Targeted drug delivery system (DDS) is required for RNA interference (RNAi) therapy to increase the therapeutic effect and to reduce the adverse effect. Especially in transthyretin (TTR)-related amyloidosis, hepatocyte specific delivery is desired because TTR mainly expresses in hepatocyte. Herein, we report on a hepatocyte-specific small interfering RNA (siRNA) delivery system using polyethylene glycol (PEG)-modified lactosylated dendrimer (generation 3; G3) conjugates with α-cyclodextrin (PEG-LαCs (G3)) for TTR-related amyloidosis therapy, and investigated the in vitro and in vivo gene silencing effect of PEG-LαCs (G3)/siRNA polyplexes. PEG-LαC (G3, average degree of substitution of PEG (DSP) 2)/TTR siRNA (siTTR) polyplex exhibited the asialoglycoprotein receptor (ASGPR)-mediated cellular uptake, high endosomal escaping ability and localization of the siRNA in cytoplasm, resulting in significant TTR silencing in HepG2 cells. In vivo studies showed that PEG-LαC (G3, DSP2)/siTTR polyplex led to a significant TTR silencing effect in liver after systemic administration to mice. Furthermore, safety evaluation revealed that PEG-LαC (G3, DSP2)/siTTR polyplex had no significant toxicity both in vitro and in vivo. These findings suggest the utility of PEG-LαC (G3, DSP2) as a promising hepatocyte-specific siRNA delivery system both in vitro and in vivo, and as a therapeutic approach for TTR-related amyloidosis.

A comprehensive understanding of lowly-hydrolyzed polyvinyl alcohol-based ternary solid dispersions with the use of a combined mixture-process design.

We recently reported lowly hydrolyzed polyvinyl alcohol (L-PVA, 70-74% hydrolyzed, about 580 polymerized, JR-05) as a promising matrix for hot-melt extrusion (HME) due to its unique micelle formation ability compared to the most commonly used PVA (87-89% hydrolyzed, about 580 polymerized). In the present study, we focused on the effect of composition [indomethacin (IND), L-PVA, sorbitol] and process parameters (temperature and screw speed) on each response, i.e. processing torque, and physicochemical properties such as residual crystallinity, residual ratio, and area under the dissolution curve (AUDC) in supersaturated solution using a HME by applying the design of experiment (DoE) approach. To overcome the poor processability of L-PVA, given its semicrystalline nature, we applied sorbitol as a plasticizer and systematically and simultaneously evaluated its influence on the outputs based on the mixture design combined with process factors. Few studies have focused on comprehensive evaluation of the composition and HME process conditions because obtaining a design space requires numerous experiments. We found that incorporating sorbitol into the L-PVA greatly improved the processing torque. However, sorbitol negatively influenced the degree of residual crystallinity and the AUDC of IND. Lastly, we established a laboratory-scale design space that could achieve high supersaturation and ensure adequate miscibility between each component, using an acceptable processing torque for HME, by applying the minimum amount of sorbitol. These fundamental results suggest that sorbitol maximizes the potency of L-PVA as a carrier in HME.

Gelation Factors of Pectin for Development of a Powder Form of Gel, Dry Jelly, as a Novel Dosage Form.

Jellies for oral administration are dosage forms that contain water, as stipulated in the Japanese Pharmacopeia, and heat is generally applied to the jellies during the manufacturing process. Therefore, it is difficult to formulate drugs that may be affected adversely by water and/or heat. To solve this problem, we tried to develop a powder form of gel as a novel dosage form (dry jelly: jelly medicine extemporaneously prepared) that is converted to jelly after addition of water at the time of administration. For this purpose, a basic gel formulation consisting of pectin, glucono-δ-lactone, dibasic calcium phosphate hydrate, and sucrose was investigated to evaluate the critical factors affecting gelation phenomena. The gel form was developed by adjusting the amount of each component of the formulation and of water added. Gelation occurred even with hard water containing metal ions (hardness of approximately 304 mg/L), and no changes in gel hardness occurred. The desired gel hardness could be controlled by adjusting the amount of water. The gel hardness changed over time after the addition of water, but this change did not affect the dissolution behavior of drugs formulated in the dry jelly.

In Vitro and In Vivo Characterization of Drug Nanoparticles Prepared Using PureNano™ Continuous Crystallizer to Improve the Bioavailability of Poorly Water Soluble Drugs.

PURPOSE: The aim of this study was to enhance the dissolution and oral absorption of poorly water-soluble active pharmaceutical ingredients (APIs) using nanoparticle suspensions prepared with a PureNano™ continuous crystallizer (PCC). METHOD: Nanoparticle suspensions were prepared with a PCC, which is based on microfluidics reaction technology and solvent-antisolvent crystallization. Phenytoin, bezafibrate, flurbiprofen, and miconazole were used as model APIs. These APIs were dissolved in ethanol and precipitated by the addition of water and polyvinyl alcohol. Batch crystallization (BC) using a beaker was also performed to prepare the suspensions. Both PCC and BC formulations were freeze-dried before being characterized in vitro and in vivo. RESULTS: The particle sizes of the nanoparticle suspensions prepared with the PCC were smaller than those prepared by BC. The dissolution rate of each API in vitro significantly increased after crystallization. Reducing the particle size of either the BC or PCC formulation led to increased API flux across Caco-2 cell monolayers. PCC preparations showed higher plasma concentrations after oral administration, demonstrating the advantages of a fast dissolution rate and increased interaction with the gastrointestinal tract owing to the smaller particle size. CONCLUSIONS: PCC can continuously produce nanoparticle APIs and is an efficient approach for improving their oral bioavailability.

Control of Drug Diffusion Behavior of Xanthan and Locust Bean Gum Gel by Agar Gel.

Oral gel formulations are known as easy to administer drug products for patients who have problems taking drugs including those with conditions such as dysphagia. In addition, there are numerous commercially available oral gel products, most of which are immediate-release formulation that release their pharmaceutical ingredient content by diffusion. This study is focused on developing oral gel formulations that reduce the dosing frequency and dosage compared to the conventional types. This is with the aim of facilitating the use of gel formulations for producing pharmaceutical agents with different dose regimens, thereby enhancing patient convenience. Here, we used naturally derived high-molecular-weight agar (Ag), xanthan gum (Xa), and locust bean gum (Lo) as gel bases to prepare a variety of gel membranes, and evaluated the diffusion coefficient of the model substances. The result revealed that the Ag content in the Xa-Lo combination gel concentration-dependently increased the diffusion coefficient. Moreover, these findings were applied in an attempt to mask the taste of intensely bitter levofloxacin. The results indicated that the Xa-Lo combination gel exhibited a significantly superior masking effect to that of the Ag gel. This study demonstrates the feasibility of using oral gel formulations to modulate the controlled-release functionality of pharmaceutical agents.

Evaluation of antitumor effects of folate-conjugated methyl-ß-cyclodextrin in melanoma.

Melanoma is a life-threatening disorder and its incidence is increasing gradually. Despite the numerous treatment approaches, conventional systemic chemotherapy has not reduced the mortality rate among melanoma patients, probably due to the induction of toxicity to normal tissues. Recently, we have developed folate-conjugated methyl-ß-cyclodextrin (FA-M-ß-CyD) and clarified its potential as a new antitumor agent involved in autophagic cell death. However, it remains uncertain whether FA-M-ß-CyD exerts anticancer effects against melanomas. Therefore, in this study, we investigated the effects of FA-M-ß-CyD on the folate receptor-α (FR-α)-expressing melanoma cell-selective cytotoxic effect. FA-M-ß-CyD showed cytotoxic effects in Ihara cells, a human melanoma cell line expressing FR-α. In sharp contrast to methyl-ß-cyclodextrin, FA-M-ß-CyD entered Ihara cells [FR-α(+)] through FR-α-mediated endocytosis. Additionally, FA-M-ß-CyD elicited the formation of autophagosomes in Ihara cells. Notably, FA-M-ß-CyD suppressed melanoma growth in BALB/c nude recombinase-activating gene-2 (Rag-2)/Janus kinase 3 (Jak3) double deficient mice bearing Ihara cells. Therefore, these results suggest that FA-M-ß-CyD could be utilized as a potent anticancer agent for melanoma chemotherapy by regulating autophagy.

Folate-appended ß-cyclodextrin as a promising tumor targeting carrier for antitumor drugs in vitro and in vivo.

A large number of antitumor drug delivery carriers based on passive targeting and/or active targeting have been developed. However, encapsulation of antitumor drugs into these drug carriers is often complicated, and antitumor activities of these targeting systems are not satisfactory. In the present study, we first prepared heptakis-6-folic acid (FA)-appended ß-cyclodextrin (ß-CyD) possessing two caproic acids between FA and a ß-CyD molecule as a spacer (Fol-c(2)-ß-CyD) and evaluated the potential as a novel tumor targeting carrier for antitumor drugs through a complexation. Fol-c(2)-ß-CyD formed an inclusion complex with doxorubicin (DOX) at a 1:1 molar ratio with a markedly high stability constant (>10(6) M(-1)). Cellular uptake of DOX was increased by the addition of Fol-c(2)-ß-CyD in KB cells, a folate receptor-α (FR-α)-positive cell line. Additionally, Fol-c(2)-ß-CyD increased in vitro antitumor activities of antitumor drugs such as DOX, vinblastine (VBL), and paclitaxel (PTX) in KB cells, but not in A549 cells, a FR-α-negative cell line. The complex of DOX with Fol-c(2)-ß-CyD markedly increased antitumor activity of DOX, not only after intratumoral administration but also after intravenous administration to mice subcutaneously inoculated Colon-26 cells, a FR-α-positive cell line. These findings suggest that Fol-c(2)-ß-CyD could be useful as a promising antitumor drug carrier.

Design and evaluation of folate-appended α-, ß-, and γ-cyclodextrins having a caproic acid as a tumor selective antitumor drug carrier in vitro and in vivo.

We reported that per-6-folic acid (FA)-appended ß-cyclodextrin (ß-CyD) possessing two caproic acids between FA and a ß-CyD molecule as a spacer (Fol-c2-ß-CyD) could be useful as a promising antitumor drug carrier. However, the effects of the cavity size and the spacer length on the carrier ability are not still known. In this study, we designed and evaluated the FA-appended three kinds of CyDs possessing a caproic acid as a spacer between FA and a CyD molecule (Fol-c1-CyDs) as a tumor targeting carrier for antitumor drugs. The stability constant of the Fol-c1-ß-CyD/doxorubicin (DOX) complex was much higher than those of Fol-c1-α-CyD and Fol-c1-γ-CyD at pH 7.3. Antitumor activity of DOX was increased by the complexation with Fol-c1-ß-CyD, but not with Fol-c1-α-CyD or Fol-c1-γ-CyD in KB cells, a folate receptor-α-positive cell line. Also, Fol-c1-ß-CyD increased antitumor activities of paclitaxel and vinblastine, but not 5-fluorouracil. Furthermore, Fol-c1-ß-CyD accelerated cellular uptake of DOX and inhibited its efflux from KB cells. The Fol-c1-ß-CyD/DOX complex showed much higher antitumor activity than DOX alone after intratumoral and intravenous administrations to tumor-bearing mice with a negligible change of the blood chemistry values. These findings suggest that Fol-c1-ß-CyD could be useful as a tumor-selective carrier for antitumor drugs.

Tannic acid-based sustained-release system for protein drugs.

In recent years, protein drug development has gained momentum, and simple and facile controlled-release systems without loss of activity are required. Herein, we developed a sustained-release system for protein drugs by exploiting the "astringency" mechanism, namely insoluble precipitate formation by interacting with tannic acid. Tannic acid formed insoluble precipitates with various protein drugs, such as nisin, insulin, lysozyme, ovalbumin, hyaluronidase, and human immunoglobulin G, through hydrophobic interactions and hydrogen bonds. The lysozyme/tannic acid complex retained in vitro lytic activity. Precipitates of the insulin/tannic acid complex prolonged hypoglycemic effects without loss of activity after subcutaneous administration. The ovalbumin/tannic acid complex enhanced anti-ovalbumin antibody production induced by ovalbumin, which may be attributed to its sustained-release profile. Accordingly, tannic acid is useful as a simple and user-friendly drug delivery system for protein drugs.

Nanoparticles of folic acid-methyl-ß-cyclodextrin (FA-MßCD)/adamantane-albumin exhibit enhanced antitumor activity compared with FA-MßCD alone.

Supramolecular drug carriers are a promising approach for delivering anticancer drugs with high blood retention after administration. We previously synthesized folic acid-modified methyl-ß-cyclodextrin (FA-MßCD) as an anticancer drug. FA-MßCD has a selective autophagy-mediated antitumor effect on folic acid receptor (FR)-expressing cancer cells. Here, we enhanced the antitumor effect and safety of FA-MßCD by preparing a supramolecular nanoparticle formulation of FA-MßCD via host-guest interactions using an adamantane conjugate with human serum albumin (Ad-HSA). The Ad-HSA/FA-MßCD supramolecular complex prolonged the blood retention of FA-MßCD and improved its antitumor effect and safety after intravenous administration in tumor-bearing mice xenografted with FR-expressing cancer cells. These results suggest that the supramolecular technique using Ad-HSA is a promising approach for the delivery of CD-based anticancer drugs.

Versatile delivery platform for nucleic acids, negatively charged protein drugs, and genome-editing ribonucleoproteins using a multi-step transformable polyrotaxane.

Various biopharmaceuticals, such as nucleic acids, proteins, and genome-editing molecules, have been developed. Generally, carriers are prepared for each biopharmaceutical to deliver it intracellularly; thus, the applications of individual carriers are limited. Moreover, the development of carriers is laborious and expensive. Therefore, in the present study, versatile and universal delivery carriers were developed for various biopharmaceuticals using aminated polyrotaxane libraries. Step-by-step and logical screening revealed that aminated polyrotaxane, including the carbamate bond between the axile molecule and endcap, is suitable as a backbone polymer. Movable and flexible properties of the amino groups modified on polyrotaxane facilitated efficient complexation with various biopharmaceuticals, such as small interfering RNA, antisense oligonucleotides, messenger RNA, ß-galactosidase, and genome-editing ribonucleoproteins. Diethylenetriamine and cystamine modifications of polyrotaxane provided endosomal-escape abilities and drug-release properties in the cytosol, allowing higher delivery efficacies than commercially available high-standard carriers without cytotoxicity. Thus, the resulting polyrotaxane might serve as a versatile and universal delivery platform for various biopharmaceuticals.

Mannose-methyl-ß-cyclodextrin suppresses tumor growth by targeting both colon cancer cells and tumor-associated macrophages.

Methylated ß-cyclodextrin (MßCD) can extract cholesterol from lipid rafts and induce apoptosis in cancer cells by inhibiting activation of the PI3K-Akt-Bad pathway. In this study, we modified MßCD with mannose (Man-MßCD) and assessed its in vitro and in vivo potential for targeting colon cancer cells expressing the mannose receptor (MR) and tumor-associated macrophages (TAM). Man-MßCD showed a significantly greater level of cellular association with colon-26 cells and M2 macrophages, and much more prominent anticancer activity than that of MßCD against MR-positive colon-26 cells. These results revealed that autophagy was the main mechanism of cell death associated with Man-MßCD. Furthermore, compared with MßCD, Man-MßCD significantly reduced tumor development following intravenous delivery to tumor-bearing mice, with no apparent side effects. Thus, Man-MßCD has the potential to be a novel anticancer drug.

Hydroxypropyl-ß-cyclodextrin Enhances Oral Absorption of Silymarin Nanoparticles Prepared Using PureNano™ Continuous Crystallizer.

The oral bioavailability of drugs is limited by factors such as poor membrane permeability, low solubility, and low dissolution rate. Silymarin (SLM) is a health-food active ingredient that is good for immunosuppression and tumor suppression. However, obtaining a good oral bioavailability is difficult owing to its poor solubility and low dissolution ability. To overcome these concerns, we previously prepared SLM nanoparticles (NPs) using the high-pressure crystallization method (PureNanoTM) and freeze-dried them with erythritol (Ery) or hydroxypropyl-ß-CyD (HP-ß-CyD) as a water-soluble dispersion stabilizer. In the present study, we investigated the mechanism underlying the improved absorption of SLM/hypromellose (HPMC)/HP-ß-CyD NPs after oral administration. The SLM/HPMC nano-suspension prepared using PureNanoTM exhibited a narrow size distribution. The size of the SLM/HPMC/HP-ß-CyD NPs was approximately 250 nm after hydration. The SLM/HPMC/HP-ß-CyD NPs were rapidly dissolved, and demonstrated a high solubility under supersaturated conditions. Additionally, they exhibited good wettability and their membrane permeability was improved compared with that of SLM original powder. These results suggest that the formulation of SLM NPs using PureNanoTM and freeze-drying with HP-ß-CyD improves the absorption of SLM after oral administration by enhancing solubility, wettability, and membrane permeability.

Design and evaluation of folate-modified liposomes for pulmonary administration in lung cancer therapy.

Pulmonary drug administration for the treatment of lung cancer is useful because the drug is directly delivered to the lung tissues with minimal invasiveness and higher efficiency compared to other conventional methods. However, it is critical to enhance drug accumulation in the lung cancer tissues to achieve sufficient therapeutic efficacy. The submicron-sized liposome (ssLip) preparation is one of the most promising approaches to enhance drug accumulation in the lungs; however, ssLips prepared for conventional inhalation do not have tumour selectivity. Therefore, in this study, we prepared folate (FA)-modified ssLip (FA-ssLip) to enhance drug accumulation in folate receptor (FR)-expressing lung cancer cells, and evaluated its physicochemical properties and potential as a drug carrier in pulmonary administration. In addition, we prepared rapamycin (RM-an autophagy-inducing anticancer drug)-loaded FA-ssLip (RM/FA-ssLip) and investigated its anti-tumour effect. FA-ssLip showed excellent nanoparticle properties with submicron size (approximately 120 nm) and high lung accumulation in lung cancer mouse model-bearing LL2 cells-a mouse Lewis lung carcinoma cell line. RM/FA-ssLip showed significant cytotoxic activity in FR-expressing cancer cells. In addition, pulmonary administration of RM/FA-ssLip extended the survival of LL2 cell tumour-bearing mice. Taken together, our results suggest the potential of FA-ssLip as a pulmonary drug carrier for the efficient treatment of lung cancer.

Polyrotaxane-Based Supramolecular Material for Improvement of Pharmaceutical Properties of Protein Drugs.

Pharmaceutical excipients, such as surfactants, amino acids, and polymers, have often been used to improve the physicochemical properties of protein drugs. However, the effects of these additives are limited because of factors such as their weak interactions with protein drugs. In the present study, we evaluated the application of a supramolecular polymer, aminated polyrotaxane (NH2-PRX), which can strongly interact with protein drugs via its dynamic and transformable properties, as a new pharmaceutical excipient for these agents. As a conventional control polymer with low mobility and average complexation ability, aminated dextran (NH2-DEX) was also prepared. NH2-PRX significantly reduced the aggregation of antibodies induced by shaking, compared with NH2-DEX. The adsorption of insulin onto glass and polypropylene containers was also reduced by the addition of NH2-PRX. In addition, the in vivo bioactivity of insulin was completely retained in the presence of NH2-PRX. Moreover, severe adverse effects were not observed following the administration of NH2-PRX. These findings indicate the potential use of NH2-PRX as a transformable pharmaceutical excipient for protein drugs.

Antigen stabilizing hydrogels based on cyclodextrins and polyethylene glycol act as type-2 adjuvants with suppressed local irritation.

Recent viral pandemics have increased global demand for vaccines. However, the supply of effective and safe vaccine not only to developed countries but also developing countries with inadequate storage equipment is still challenging due to the lack of robust systems which improve the efficacy and the stability of vaccines with few side effects. In our previous study, polypseudorotaxane (PPRX) hydrogels based on cyclodextrin (CyD) and polyethylene glycol (PEG) significantly improved the stability of antibody preparations and showed no serious adverse effects after subcutaneous injection, suggesting the possibility as safe vaccine formulations to stabilize an antigen protein. Moreover, recent studies have reported that one of the CyD derivatives, hydroxypropyl-ß-CyD (HP-ß-CyD), acts as an adjuvant to enhance protective type-2 immune responses. However, it is still unknown that CyD PPRX hydrogels enhance not only the stability of an antigen protein but also its immunogenicity with tolerable side effects. Here, we demonstrate that α- and γ-CyD PPRX hydrogels containing an antigen protein significantly induce antigen-specific type-2 immune responses. Moreover, α- and γ-CyD PPRX hydrogels showed negligible local irritation at the injection site, although subcutaneous injection of α-CyD alone induced skin lesion. Finally, shaking stability of the antigen protein at room temperature was significantly improved by being included in α- and γ-CyD PPRX hydrogels. These results propose the possibility of α- and γ-CyD PPRX hydrogels as novel vaccine formulations which improve both the immunogenicity and stability of an antigen protein with suppressed local irritation.

Stabilization and Movable Ligand-Modification by Folate-Appended Polyrotaxanes for Systemic Delivery of siRNA Polyplex.

To achieve a systemic targeted delivery of siRNA using polymeric carriers, there is a dilemma between ligand modification and stabilization of the polyplex. Namely, ligand modification often leads to destabilization of the polyplex in the blood circulation. In fact, we previously developed cyclodextrin (CD)/polyamidoamine dendrimer conjugates (CDE) as siRNA carriers, and the interaction of CDE/siRNA was decreased by the conjugation with folate-polyethylene glycol, leading to the destabilization. To overcome this dilemma, in this study, folate-appended polyrotaxanes (Fol-PRX) were developed. Fol-PRX stabilized CDE/siRNA polyplex by intermolecularly connecting CDE molecules through a host-guest interaction between adamantane at the terminals of Fol-PRX and ß-CD in the polyplex. Moreover, the intermolecular connection of the polyplex with Fol-PRX provided movable folate moieties on the surface. As a result, Fol-PRXs enhanced the in vivo antitumor activity of the polyplex after intravenous administration, suggesting their utility as the dual-functional materials for systemic delivery of siRNA polyplexes.