Folate-Appended Hydroxypropyl-ß-Cyclodextrin Induces Autophagic Cell Death in Acute Myeloid Leukemia Cells.

Acute myeloid leukemia (AML) is a heterogenous myeloid neoplasm that remains challenging to treat. Because intensive conventional chemotherapy reduces survival rates in elderly patients, drugs with lower toxicity and fewer side effects are needed urgently. 2-Hydroxypropyl-ß-cyclodextrin (HP-ß-CyD) is used clinically as a pharmaceutical excipient for poorly water-soluble drugs. Previously, we showed that HP-ß-CyD exerts antitumor activity by disrupting cholesterol homeostasis. Recently, we developed folate-conjugated HP-ß-CyD (FA-HP-ß-CyD) and demonstrated its potential as a new antitumor agent that induces not only apoptosis, but also autophagic cell death; however, we do not know whether FA-HP-ß-CyD exerts these effects against AML. Here, we investigated the effects of FA-HP-ß-CyD on folate receptor (FR)-expressing AML cells. We found that the cytotoxic activity of FA-HP-ß-CyD against AML cells was stronger than that of HP-ß-CyD. Also, FA-HP-CyD induced the formation of autophagosomes in AML cell lines. FA-HP-ß-CyD increased the inhibitory effects of cytarabine and a BCL-2-selective inhibitor, Venetoclax, which are commonly used treat elderly AML patients. Notably, FA-HP-ß-CyD suppressed the proliferation of AML cells in BALB/c nude recombinase-activating gene-2 (Rag-2)/Janus kinase 3 (Jak3) double-deficient mice with AML. These results suggest that FA-HP-ß-CyD acts as a potent anticancer agent for AML chemotherapy by regulating autophagy.

Different solubilizing ability of cyclodextrin derivatives for cholesterol in Niemann-Pick disease type C treatment.

BACKGROUND: Niemann-Pick disease type C (NPC) is a fatal neurodegenerative disorder caused by abnormal intracellular cholesterol trafficking. Cyclodextrins (CDs), the most promising therapeutic candidates for NPC, but with concerns about ototoxicity, are cyclic oligosaccharides with dual functions of unesterified cholesterol (UC) shuttle and sink that catalytically enhance the bidirectional flux and net efflux of UC, respectively, between the cell membrane and the extracellular acceptors. However, the properties of CDs that regulate these functions and how they could be used to improve treatments for NPC are unclear. METHODS: We estimated CD-UC complexation for nine CD derivatives derived from native α-, ß-, and γ-CD with different cavity sizes, using solubility and molecular docking analyses. The stoichiometry and complexation ability of the resulting complexes were investigated in relation to the therapeutic effectiveness and toxicity of each CD derivative in NPC experimental models. FINDINGS: We found that shuttle and sink activities of CDs are dependent on cavity size-dependent stoichiometry and substituent-associated stability of CD-UC complexation. The ability of CD derivatives to form 1:1 and 2:1 complexes with UC were correlated with their ability to normalize intracellular cholesterol trafficking serving as shuttle and with their cytotoxicity associated with cellular UC efflux acting as sink, respectively, in NPC model cells. Notably, the ability of CD derivatives to form an inclusion complex with UC was responsible for not only efficacy but ototoxicity, while a representative derivative without this ability negligibly affected auditory function, underscoring its preventability. CONCLUSIONS: Our findings highlight the importance of strategies for optimizing the molecular structure of CDs to overcome this functional dilemma in the treatment of NPC.

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.

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.

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.

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.

Fine-tuned cholesterol solubilizer, mono-6-O-α-D-maltosyl-γ-cyclodextrin, ameliorates experimental Niemann-Pick disease type C without hearing loss.

Niemann-Pick disease type C (NPC) is a fatal disorder with abnormal intracellular cholesterol trafficking resulting in neurodegeneration and hepatosplenomegaly. A cyclic heptasaccharide with different degrees of substitution of 2-hydroxypropyl groups, 2-hydroxypropyl-ß-cyclodextrin (HP-ß-CD), acts as a strong cholesterol solubilizer and is under investigation for treating this disease in clinical trials, but its physicochemical properties and ototoxicity remain a concern. Here, we evaluated the potential of mono-6-O-α-maltosyl-γ-CD (G2-γ-CD), a single-maltose-branched cyclic octasaccharide with a larger cavity than HP-ß-CD, for treating NPC. We identified that G2-γ-CD ameliorated NPC manifestations in model mice and showed lower ototoxicity in mice than HP-ß-CD. To investigate the molecular mechanisms of action behind the differential ototoxicity of these CDs, we performed cholesterol solubility analysis, proton nuclear magnetic resonance spectroscopy, and molecular modeling, and estimated that the cholesterol inclusion mode of G2-γ-CD maintained solely the 1:1 inclusion complex, whereas that of HP-ß-CD shifted to the highly-soluble 2:1 complex at higher concentrations. We predicted the associations of these differential complexations of CDs with cholesterol with the profile of disease attenuation and of the auditory cell toxicity using specific cell models. We proposed that G2-γ-CD can serve as a fine-tuned cholesterol solubilizer for treating NPC, being highly biocompatible and physicochemically suitable for clinical application.

Multifunctional Therapeutic Cyclodextrin-Appended Dendrimer Complex for Treatment of Systemic and Localized Amyloidosis.

Amyloidosis pathologically proceeds via production of amyloidogenic proteins by organs, formation of protein aggregates through structural changes, and their deposition on tissues. A growing body of evidence demonstrates that amyloidosis generally develops through three critical pathological steps: (1) production of amyloid precursor proteins, (2) amyloid formation, and (3) amyloid deposition. However, no clinically effective therapy that is capable of targeting each pathological step of amyloidosis independently is currently available. Here, we combined therapeutic effects and developed a short hairpin RNA expression vector (shRNA) complex with a cyclodextrin-appended cationic dendrimer (CDE) as a novel multitarget therapeutic drug that is capable of simultaneously suppressing these three steps. We evaluated its therapeutic effects on systemic transthyretin (ATTR) amyloidosis and Alzheimer's disease (AD) as localized amyloidosis, by targeting TTR and amyloid ß, respectively. CDE/shRNA exhibited RNAi effects to suppress amyloid protein production and also achieved both inhibition of amyloid formation and disruption of existing amyloid fibrils. The multitarget therapeutic effects of CDE/shRNA were confirmed by evaluating TTR deposition reduction in early- and late-onset human ATTR amyloidosis model rats and amyloid ß deposition reduction in AppNL-G-F/NL-G-F AD model mice. Thus, the CDE/shRNA complex exhibits multifunctional therapeutic efficacy and may reveal novel strategies for establishing curative treatments for both systemic and localized amyloidosis.

"Human and Mouse Cross-Reactive" Albumin-Binding Helix-Loop-Helix Peptide Tag for Prolonged Bioactivity of Therapeutic Proteins.

The effectiveness of protein and peptide pharmaceuticals depends essentially on their intrinsic pharmacokinetics. Small-sized pharmaceuticals in particular often suffer from short serum half-lives due to rapid renal clearance. To improve the pharmacokinetics by association with serum albumin (SA) in vivo, we generated an SA-binding tag of a helix-loop-helix (HLH) peptide to be linked with protein pharmaceuticals. For use in future preclinical studies, screening of yeast-displayed HLH peptide libraries against human SA (HSA) and mouse SA (MSA) was alternately repeated to give the SA-binding peptide AY-VE, which exhibited cross-binding activities to HSA and MSA with KD of 65 and 20 nM, respectively. As a proof of concept, we site-specifically conjugated peptide AY-VE with insulin to examine its bioactivity in vivo. In mouse bioassay monitoring the blood glucose level, the AY-VE conjugate was found to have a prolonged hypoglycemic effect for 12 h. The HLH peptide tag is a general platform for extending the bioactivity of therapeutic peptides or proteins.

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.

Folic Acid-Appended Hydroxypropyl-ß-Cyclodextrin Exhibits Potent Antitumor Activity in Chronic Myeloid Leukemia Cells via Autophagic Cell Death.

2-Hydroxypropyl-ß-cyclodextrin (HP-ß-CyD) is widely used as an enabling excipient in pharmaceutical formulations. We previously demonstrated that HP-ß-CyD disrupted cholesterol homeostasis, and inhibited the proliferation of leukemia cells by inducing apoptosis and cell-cycle arrest. Recently developed drug delivery systems using folic acid (FA) and folic acid receptors (FR) are currently being used in cancer treatment. To confer tumor cell-selectivity to HP-ß-CyD, we synthesized folate-appended HP-ß-CyD (FA-HP-ß-CyD) and evaluated the potential of FA-HP-ß-CyD as an anticancer agent using chronic myeloid leukemia (CML) cells in vitro and in vivo. FA-HP-ß-CyD inhibited the growth of FR-expressing cells but not that of FR-negative cells. FA-HP-ß-CyD had stronger anti-leukemia and cell-binding activities than HP-ß-CyD in CML cells. Unlike HP-ß-CyD, FA-HP-ß-CyD entered CML cells through endocytosis and induced both apoptosis and autophagy via mitophagy. FA-HP-ß-CyD increased the inhibitory effects of the ABL tyrosine kinase inhibitors imatinib mesylate and ponatinib, which are commonly used in CML. In vivo experiments in a BCR-ABL leukemia mouse model showed that FA-HP-ß-CyD was more effective than HP-ß-CyD at a ten-fold lower dose. These results indicate that FA-HP-ß-CyD may be a novel tumor-targeting agent for the treatment of leukemia.

Computational Screening of a Functional Cyclodextrin Derivative for Suppressing a Side Effect of Doxorubicin.

The binding affinity of the beta-cyclodextrin (ß-CyD) derivatives with Doxorubicin (Dox) is evaluated by means of the 3D-RISM/KH theory combined with the molecular dynamics simulation in order to screen the compounds for suppressing a side-effect of the cancer drug. A protocol revised for the external and conformational entropies of the host-guest system is employed to calculate the binding free energy. It is found that the direct interactions of CyD with Dox and the desolvation free-energies of the both compounds largely cancel out to leave moderate contributions to the affinity, which are comparable to those from the entropies. The results shed light on the entropy terms for determining the binding affinity, although the external-entropy terms are essentially constant over all the compounds examined and do not affect the screening. The theoretical result is compared with the experimental data of the association constant for a CyD derivative which was predicted to be the best compound by the preliminary calculation without the entropy terms.

Polypseudorotaxane-based supramolecular hydrogels consisting of cyclodextrins and Pluronics as stabilizing agents for antibody drugs.

Recently, antibody drugs have been used worldwide, and based on worldwide sales, 7 of the top 10 pharmaceutical products in 2019 were antibody-based drugs. However, antibody drugs often form aggregates upon thermal and shaking stresses with few efficient stabilizing agents against both stresses. Herein, we developed polypseudorotaxane (PpRX) hydrogels consisting of cyclodextrins (CyDs) and polyethylene glycol (PEG)-polypropylene glycol (PPG)-PEG block copolymers (Pluronics F108, F87, F68, and L44), and evaluated their utility as antibody stabilizing agents. α- and γ-CyDs formed PpRX hydrogels with Pluronics, where CyD/F108 gels showed remarkable stabilizing effects for human immunoglobulin G (IgG) against both thermal and shaking stresses beyond CyD/PEG gels or generic gels. The effects were probably due to the interaction between IgG and the free PPG block of Pluronic F108, resulting in the strong IgG retention in the gels. These findings suggest the great potential of CyD/Pluronic gels as pharmaceutical materials for antibody formulations.

Sacran, a sulfated polysaccharide, suppresses the absorption of lipids and modulates the intestinal flora in non-alcoholic steatohepatitis model rats.

AIMS: The objective of this study was to investigate the effects of administering sacran, a sulfated polysaccharide, on liver biology, gut microbiota, oxidative stress, and inflammation on stroke-prone spontaneously hypertensive (SHRSP5/Dmcr) rats that develop fibrotic steatohepatitis with histological similarities to that of non-alcoholic steatohepatitis (NASH). MAIN METHODS: Four groups of 8-week-old SHRSP5/Dmcr rats were fed a high fat-cholesterol (HFC) diet for 4 and 8 weeks and administered either sacran (80 mg/kg/day) or a non-treatment, respectively. Liver function was evaluated by biochemical and histopathological analyses. Hepatic inflammatory markers were measured using mRNA expression. Fecal microbial profiles were determined via 16S rRNA sequencing. A triglyceride (TG) absorption test was administered to the 8-week-old Sprague-Dawley (SD) rats. KEY FINDING: Sacran administration was observed to decrease the extent of oxidative stress and hepatic biochemical parameters in serum and hepatic injury with the levels of transforming growth factor-beta (TGF-ß1) and tumor necrosis factor-alpha (TNF-α), being increased compared to those of the non-treatment group. At the genus level, sacran administration caused a significant decrease in the harmful Prevotella genus, and a significant increase in the useful Blautia genus was observed. Sacran administration also decreased the serum TG increase that was induced by administering corn oil to the SD rats. SIGNIFICANCE: We conclude that sacran administration has the potential to reduce the absorption of lipids into blood and to improve several gut microbiotas, in the gastrointestinal tract, thereby inhibiting the subsequent development of oxidative stress and hepatic markers in the systematic circulation on NASH.

Intracerebroventricular Treatment with 2-Hydroxypropyl-ß-Cyclodextrin Decreased Cerebellar and Hepatic Glycoprotein Nonmetastatic Melanoma Protein B (GPNMB) Expression in Niemann-Pick Disease Type C Model Mice.

Niemann-Pick disease type C (NPC) is a recessive hereditary disease caused by mutation of the NPC1 or NPC2 gene. It is characterized by abnormality of cellular cholesterol trafficking with severe neuronal and hepatic injury. In this study, we investigated the potential of glycoprotein nonmetastatic melanoma protein B (GPNMB) to act as a biomarker reflecting the therapeutic effect of 2-hydroxypropyl-ß-cyclodextrin (HP-ß-CD) in an NPC mouse model. We measured serum, brain, and liver expression levels of GPNMB, and evaluated their therapeutic effects on NPC manifestations in the brain and liver after the intracerebroventricular administration of HP-ß-CD in Npc1 gene-deficient (Npc1-/-) mice. Intracerebroventricular HP-ß-CD inhibited cerebellar Purkinje cell damage in Npc1-/- mice and significantly reduced serum and cerebellar GPNMB levels. Interestingly, we also observed that the intracerebral administration significantly reduced hepatic GPNMB expression and elevated serum ALT in Npc1-/- mice. Repeated doses of intracerebroventricular HP-ß-CD (30 mg/kg, started at 4 weeks of age and repeated every 2 weeks) drastically extended the lifespan of Npc1-/- mice compared with saline treatment. In summary, our results suggest that GPNMB level in serum is a potential biomarker for evaluating the attenuation of NPC pathophysiology by intracerebroventricular HP-ß-CD treatment.

Improvement of Pharmaceutical Properties of Zerumbone, a Multifunctional Compound, Using Cyclodextrin Derivatives.

Zerumbone is a multifunctional compound which shows various biological activities, such as antitumor activity, anti-inflammatory activity, antiulcer activity, etc. However, to use Zerumbone as functional foods or medicines, its pharmaceutical properties such as solubility should be improved. In the present study, we prepared its inclusion complexes with various cyclodextrin (CyD) derivatives, and evaluated their solubility, release profile of the drug and cytotoxic activity. Among 11 CyDs, sulfobutylether (SBE)-ß-CyD showed the highest solubilizing effect for Zerumbone. Phase solubility diagrams of SBE-ß-CyD/Zerumbone in 10% methanol solution showed AL type, and the stability constant was 756 M-1. SBE-ß-CyD also formed the solid complex with Zerumbone by kneading for 90 min. Importantly, the dissolution rate of Zerumbone was improved by complexation with SBE-ß- and hydroxypropyl (HP)-ß-CyDs, and its supersaturation was maintained for several hours. The solubilizing effects by SBE-ß-CyD was greater than that of HP-ß-CyD. Moreover, SBE-ß-CyD/Zerumbone complex also retained the cytotoxic activity of Zerumbone. These results suggest that CyDs, especially SBE-ß-CyD, were useful to improve the solubility of Zerumbone.

Folate-appended cyclodextrin improves the intratumoral accumulation of existing boron compounds.

In this study, the tumor accumulation and antitumor effect of folate-modified cyclodextrin (ND201) purified with folate receptor (FR) connotated with BSH were examined. ND201 and BSH were stably bound in blood, and the mixing ratio 1:1 was most efficient. ND-BSH showed higher boron concentration (38.5 ppm) than BSH alone (11.25 ppm). The maximum ND-BSH tumor/blood ratio was also markedly higher (6.58) than that of BSH alone (1.04). ND-BSH showed a significant antitumor effect compared with BSH after neutron irradiation.

Genome Editing in a Wide Area of the Brain Using Dendrimer-Based Ternary Polyplexes of Cas9 Ribonucleoprotein.

A preassembled Cas9/single-guide RNA complex (Cas9 ribonucleoprotein; Cas9 RNP) induces genome editing efficiently, with small off-target effects compared with the conventional techniques, such as plasmid DNA and mRNA systems. However, penetration of Cas9 RNP through the cell membrane is low. In particular, the incorporation of Cas9 RNP into neurons and the brain is challenging. In the present study, we have reported the use of a dendrimer (generation 3; G3)/glucuronylglucosyl-ß-cyclodextrin conjugate (GUG-ß-CDE (G3)) as a carrier of Cas9 RNP and evaluated genome editing activity in the neuron and the brain. A Cas9 RNP ternary complex with GUG-ß-CDE (G3) was prepared by only mixing the components. The resulting complex exhibited higher genome editing activity than the complex with the dendrimer (G3), Lipofectamine 3000 or Lipofectamine CRISPRMAX in SH-SY5Y cells, a human neuroblastoma cell line. In addition, GUG-ß-CDE (G3) enhanced the genome editing activity of Cas9 RNP in the whole mouse brain after a single intraventricular administration. Thus, GUG-ß-CDE (G3) is a useful Cas9 RNP carrier that can induce genome editing in the neuron and brain.