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Nonpolymer, pH-sensitive carbon dots (pSCDs) were developed to overcome the disadvantages of pH-sensitive polymers such as inevitable synthesis, wide distribution of molecular weight, uncontrolled loading and release rate of drugs, and toxicity by biodegradation. The pSCDs were synthesized via one spot synthesis for 3 min using citric acid (CA) and 1-(3-aminopropyl) imidazole (API). Imidazole groups were present on pSCD surfaces and facilitated DOX loading via hydrophobic interactions (loading efficiency: 78.55%). The DOX-loaded pSCDs collapsed at tumoral pH (pH ∼ 6.5) due to protonation of the imidazole groups, and DOX was released about 7 times higher than the control group. The therapeutic effect was confirmed in vitro using HCT-116 (human colon cancer), PANC-1 (human pancreatic cancer), and SKBR-3 (human breast cancer) cells. Additionally, the DOX-loaded pSCDs successfully inhibited tumor growth in an HCT-116-bearing mouse model and did not show toxicity. These results indicate that a nonpolymeric pSCDs platform has the potential to be used as a cancer targeting therapeutic material.

A combination of chemo-photodynamic therapy has been manifested as a promising strategy for efficient cancer treatment due to the enhanced therapeutic efficacy. Here, we designed doxorubicin (DOX)-loaded photoresponsive micelles (DPRMs) based on a combination of chlorin e6 (Ce6) and lipoic acid (LA) conjugated methoxy-poly(ethylene) glycol (mPEG-Ce6, mPEG-LA) to achieve effective drug delivery using a single system. DPRMs were optimized with different molar ratios of mPEG-Ce6 and mPEG-LA which showed uniformly spherical morphology of size ∼130 nm and approximately 9% of DOX loading contents. Photoresponsive lipoyl ring of mPEG-LA was incorporated in DPRMs in order to induce photomediated reduction resulting in 2-3-fold accelerated DOX release according to higher molar ratio of mPEG-LA and enhancement of light dose. The photoresponsive DOX release and ROS generation by Ce6 mediated cytotoxic effect of DPRMs were demonstrated in vitro using CT-26 (mouse colon cancer) and HCT-116 (human colon cancer) cells. We observed both the photosensitizer and the anticancer drug are colocalized in the tumor cells to achieve effective enhancement. Additionally, the DPRMs with laser irradiation successfully inhibited tumor growth in CT-26 tumor bearing mouse model and immunohistochemical staining verified apoptosis-mediated tumor growth inhibition. These observations demonstrated that the DPRMs showed a higher therapeutic effect than the other systems and PDT maximized the antitumor effect. Thus, DPRMs confirmed the advantages as a chemo-photodynamic dual-therapy with a synergistic therapeutic effect and great potential for cancer treatment.

To overcome the limitations of single therapy, chemotherapy has been studied to be combined with photodynamic therapy. However, nanomedicine combining anticancer drug and photosensitizer still cannot address the insufficiency of drug delivery and the off-targeting effect. To address drug delivery issue, we have developed a doxorubicin encapsulating human serum albumin nanoparticles/chlorin e6 encapsulating microbubbles (DOX-NPs/Ce6-MBs) complex system. Microbubbles enable ultrasound-triggered local delivery via sonoporation for maximizing the drug delivery to a target site. In both in vitro and in vivo experiments, the developed DOX-NPs/Ce6-MBs drug delivery complex could be confirmed to transfer drugs deeply and effectively into cancerous tumors through the following three steps; (1) the local release of nanoparticles due to the cavitation of DOX-NPs/Ce6-MBs; (2) the enhanced extravasation of DOX-NPs and Ce6-liposome/micelle due to the sonoporation phenomenon; (3) the improved penetration of extravasated nanomedicines into the deep tumor region due to the mechanical energy of ultrasound. As a result, the developed DOX-NPs/Ce6-MBs complex with ultrasound irradiation showed increased therapeutic effects compared to the case where no ultrasound irradiation was applied. The DOX-NPs/Ce6-MBs was concluded from this study to be the optimal drug delivery system for external-stimuli local combination (chemotherapy + PDT) therapy., (Copyright © 2018 Elsevier B.V. All rights reserved.)

To develop a reactive oxygen species (ROS) sensitive drug carrier, a chondroitin sulfate (CS)-anthocyanin (ATC) based nanocomplex was developed. Doxorubicin hydrochloride (DOX) was loaded in the CS-ATC nanocomplex (CS-ATC-DOX) via intermolecular stacking interaction. The nanocomplex was fabricated by a simple mixing method in the aqueous phase. The morphology and size of CS-ATC-DOX were determined by ATC content. In the group with 1.5mg/ml of ATC loaded CS-ATC-DOX (CS-ATC2-DOX), the drug content and loading efficiency were 8.5% and 99.1%, respectively. The ROS sensitive drug release of CS-ATC2-DOX was confirmed under in vitro physiological conditions. The results demonstrated that 1.67 times higher DOX release occurred in CS-ATC2-DOX for 48h compared to CS-DOX (ATC absent sample). Drug release and nanocomplex destruction were induced by ROS mediated ATC degradation. We determined that 66.7% of ROS was scavenged by CS-ATC2-DOX. Additionally, an HCT-116 tumor bearing animal model was used to confirm ROS sensitive therapeutic effects of CS-ATC2-DOX. The results indicate that DOX was released from the intravenously injected CS-ATC2-DOX in the tumor tissue. Thus, nuclei shrinkage and dead cells were observed in H&E staining and TUNEL assay, respectively. These data suggest that the tumor growth was effectively inhibited. This study means that CS-ATC2-DOX has potential in improving tumor therapy., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Light-triggered drug delivery is among the most investigated stimulus-response strategies and has been widely explored in cancer treatment. However, the limited specificity of light-triggered drug delivery reduces the therapeutic efficacy and causes considerable undesirable side effects. In this work, we demonstrate a highly tumor-specific light-triggerable drug carrier (H-LTDC) induced by a combination of internal (i.e., tumor hypoxia) and external stimuli (i.e., light). The doxorubicin (DOX)-loaded H-LTDC was self-assembled from type-1-reactive oxygen species (ROStype1)-mediated degradable chondroitin sulfate (CS) conjugated with a photosensitizer (PS), Pheophorbide-a, which has a spherical shape and a uniform size distribution. Under hypoxic conditions, ROSType1 was mainly generated due to the electron-rich sulfate groups in the polysaccharide backbone. The ROStype1 generated by H-LTDC allowed laser-triggered drug release at low oxygen concentrations. From the in vitro cytotoxicity tests with colon cancer cells (HCT-116), under laser irradiation, DOX-loaded H-LTDCs showed higher toxicity under hypoxic conditions than that under normoxic conditions. In vivo and ex vivo biodistribution studies demonstrated that H-LTDCs selectively accumulated in the tumor tissues. As a result, drug-loaded H-LTDCs exhibited high anti-tumor activity in vivo. Overall, we believe that this approach could represent a promising platform for the treatment of tumor and hypoxia-associated diseases without undesirable side effects., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Nanoparticles with "smart" stimuli-responsive materials and multiple therapeutic strategies in a single delivery platform have emerged for highly efficient cancer therapy. Here, photomediated reactive oxygen species (ROS)-generable nanoparticles are designed that can trigger drug release and endo/lysosomal escape upon attenuated single light irradiation, simultaneously, for synergistic chemo-photodynamic ablation. In this study, the self-ROS-generable nanoparticles (SRNs) are prepared from the polymer based on polysaccharide, chlorin e6 as ROS generator and lipoic acid as ROS scavenger covalently conjugated pullulan with anticancer drug (doxorubicin, DOX) through self-assembly, and can disassemble via the ROS-mediated reduction of lipoyl group in response to low level exogenous single light switch. After cellular internalization in hepatic cancer through asialoglycoprotein receptor (ASGPR, as pullulan receptor)-mediated endocytosis, once irradiated, SRNs are able to produce ROS that can simultaneously induce drug release triggering and endo/lysosomal escape of DOX into cytoplasm as well as directly photodynamic therapy for highly efficient chemo-photodynamic cancer therapy. This promising delivery system, which has huge potential in biomedical applications, may be optimal for smart delivery platform., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Drug resistance remains one of the primary obstacles to the success of cancer chemotherapy. In this work, we demonstrate a singlet-oxygen producible polymeric (SOPP) micelle based on photosensitizer (PS, chlorin e6 (Ce6)) conjugated amphiphilic copolymer (pluronic F127(®), PF127) for overcoming drug resistance in cancer by applying photochemical internalization (PCI). The doxorubicin (DOX)-loaded SOPP micelles were self-assembled from Ce6-PF127 conjugates, which have a spherical shape with a uniform size of ∼30 nm. Compared with free Ce6, enhanced singlet-oxygen generation efficiency in the DOX-loaded SOPP micelles have been demonstrated in aqueous environments due to their increased water-dispersibility. Under low dose of laser power and anti-cancer drug (DOX) conditions, in vitro and in vivo studies on drug-resistant cancer cells demonstrated that singlet-oxygen-mediated cellular membrane damage (caused by lipid peroxidation) significantly increased the cellular uptake of drug (DOX), which led to overcoming the drug resistance in cancer cells without undesirable side effects. We believe this approach could represent a promising platform for drug-resistant cancer treatment., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

In order to develop immuno- and chemotherapy agents, self-organized acetylated fucoidan (AcFu) nanoparticles were designed. Doxorubicin (DOX), used as a model drug, was loaded into the AcFu nanoparticles by dialysis. The DOX loading efficacy and content were 71.1% and 3.6%, respectively. Approximately 140 nm of spherical nanoparticles were obtained. DOX-loaded AcFu nanoparticles (DOX-AcFu) exhibited first-order drug release behavior for 5 days. Interestingly, AcFu treated Raw264.7 macrophages overexpressed various anti-tumor cytokines, such as tumor necrosis factor-alpha (TNF-α) and granulocyte-macrophage colony-stimulating factor (GM-CSF). The ability of DOX-AcFu to suppress drug efflux was revealed by confocal microscope images and FACS analysis in multidrug resistance (MDR) cells. IC50 (50% inhibitory concentration) value of DOX-AcFu was lower than that of free DOX in the MDR model cells. Based on these results, we strongly suggest that AcFu nanoparticles have a promising potential for development as a one-step therapy containing agents for both immuno- and chemotherapy., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Limited distribution of anticancer drugs has been recognized as a significant hurdle to efficacy. We investigated a detailed penetration/distribution profile of paclitaxel-rhodamine (PTX-rd) and doxorubicin (DOX) in multicellular layer (MCL) cultures of human cancer cells as an in vitro model for avascular regions of solid tumors. MCLs were exposed to drugs and fluorescent images of frozen sections were acquired for determination of drug penetration into MCL under various exposure conditions. PTX-rd and DOX showed drastically different profiles of penetration. DOX showed full penetration after 1 h and accumulation over 3 h, whereas PTX-rd showed slow and limited penetration, with accumulation only within the top 20% of layers by 2 h and insignificant penetration even at 72 h. Drug retention in MCL was more dependent on drug concentration, rather than exposure time, i.e., drug distribution increased by 6.3- and 2.5-fold for PTX-rd and DOX, respectively, when exposed to higher concentrations under comparable AUC exposure (1 µM x 24 h vs. 50 µM x 0.5 h). Anti-proliferative activity of PTX and DOX in MCL, as determined by cell cycle analysis, was minimal and may be attributed, at least in part, to their limited distribution in multicellular cultures. Overall, we demonstrated that penetration and retention of PTX and DOX in MCL was not only concentration- and time-dependent, but also schedule-dependent. It is suggested that slow releasing formulations or a slow infusion regimen may not necessarily be desirable, especially for PTX, due to insufficient penetration and accumulation which may result from a low local concentration at the target site.

The principal objective of this study was to fabricate doxorubicin-loaded self-organized nanogels composed of hydrophobized pullulan (PUL)-Nalpha-Boc-L-histidine (bHis) conjugates. Their responses to tumor extracellular pH (pHe) were determined, and they were also evaluated with regard to their anticancer efficacy against breast cancer cell lines (MCF-7). bHis was grafted to a PUL-deoxycholic acid (DO) conjugate (PUL-DO) via an ester linkage. PUL-DO/bHis conjugates with two different degrees of bHis substitutions (PUL-DO/bHis36 and PUL-DO/bHis78) were synthesized. PUL-DO/bHis nanogels formed via dialysis at a pH of 8.5 evidenced larger particle sizes (<140 nm) and lower critical aggregation concentrations (CACs) than did the PUL-DO nanogels (90 nm). The pH-dependent CAC of PUL-DO/bHis78 changed dramatically, from 1.2 microg/mL at pH 8.5, to 10 at 7.0, and to 660 at 6.2. A similar tendency in pH-dependent size was also noted. The ionization of the imidazole ring in bHis is principally responsible for pH dependency. The bHis moieties function as a switching tool responding to external pH. Doxorubicin (DOX)-loaded nanogels were assessed for pH-dependent releasing kinetics. The release rate of DOX from the PUL-DO/bHis78 nanogels increased significantly with reductions in pH. This resulted in increased cytotoxicity (30% cell viability at a dose of 10 microg/mL DOX equivalent) against sensitive MCF-7 cells at a pH of 6.8 and low cytotoxicity at pH 7.4 (65% cell viability at an identical dose). The results show that PUL-DO/bHis nanogels may potentially be employed as anti-tumor drug carriers.

In order to overcome multidrug resistance in solid tumors, doxorubicin (DOX) loaded pH-sensitive micelles of which surface was decorated with folate (PHSM/f) were evaluated both in vitro and in vivo experiments. PHSM/f were fabricated from a mixture of two block copolymers of poly(L-histidine) (M(n): 5K)-b-PEG (M(n): 2K)-folate (polyHis/PEG-folate) (75 wt.%) and poly(L-lactic acid) (M(n): 3K)-b-PEG (M(n): 2K)-folate (PLLA/PEG-folate) (25 wt.%). The PHSM/f showed more than 90% cytotoxicity of DOX resistant MCF-7 (MCF-7/DOX(R)) when cultured with PHSM/f at a concentration of 10 microg/ml DOX. The result was interpreted by a sequential event of active internalization of PHSM/f via folate-receptor mediated endocytosis and ionization of His residues which result in micelle destabilization and probably disturbance of endosomal membranes. This potential mechanism may endow the drug carriers to bypass Pgp efflux pump and sequestration of DOX in acidic intracellular compartments, yielding high cytotyoxicity. Experimental evaluation of tumor regression was carried out in a small animal model bearing s.c. MCF-7 or MCF-7/DOX(R) xenografts. The tumor (MCF-7/DOX) volumes of mice treated with PHSM/f were significantly less than control groups treated with free DOX or similar micelles but without folate (PHSM). In the MCF-7/DOX(R) xenograft model, the accumulated DOX level of PHSM/f in solid tumors was 20 times higher than free DOX group, and 3 times higher than PHSM group. The results demonstrate that PHSM/f is a viable means for treating drug resistant tumors.

The cytotoxicity of adriamycin (ADR)-loaded and pH-sensitive nanoparticles made of pullulan acetate (PA) and sulfonamide (sulfadimethoxine; SDM) (PA/SDM) conjugate to a breast tumor cell line (MCF-7) was investigated to test the feasibility of the nanoparticles in targeting acidic tumor extracellular pH (pH(e)). At pH 6.8, ADR loaded PA/SDM nanoparticles showed cytotoxicity in the cell culture experiment, comparable to that of free ADR at the same ADR concentrations, while the relative cytotoxicity at pH 7.4 was low at the tested concentration range. This pronounced cytotoxicity of the nanoparticles at low pH was attributed to the accelerated release of ADR triggered by pH, enhanced interaction with cells, and internalization. At pH 6.8 and 6.4, the PA/SDM nanoparticles aggressively bounded to MCF-7 cells, probably due to interactions of the cells with hydrophobized nanoparticle surfaces caused by SDM deionization. A confocal laser microscopic study revealed intracellular localization of the drug-loaded nanoparticles. Based on these findings, the pH-sensitive nanoparticles deserve further investigation with an in vivo animal model as a targeted carrier of pH(e)., (Copyright 2002 Elsevier Science B.V.)

Much progress has been made toward stimuli‐responsive polysaccharide‐based selective tumor therapy not only because polysaccharides have nontoxic biodegradability and biocompatibility but also because their stimuli‐sensitive characteristics enable the proper transport of payloads into tumors. Here, we attempted to deliver an antitumor drug, doxorubicin (DOX), using starch‐based microparticles coupled with pH‐responsive 3‐(diethylamino)propylamine. The microparticles of starch conjugated with 3‐(diethylamino)propylamine (SDEAP) allowed for the change in hydrophobicity of SDEAPs in a pH‐dependent manner. The results revealed that SDEAPs effectively carried and released DOX and selectively killed tumor cells under acidic condition. Overall, this study suggests that DOX‐loaded SDEAPs can be further explored as a strategy for applications to acidic tumor‐targeting implants owing to the drug‐deliver efficiency and tumor selectivity. [ABSTRACT FROM AUTHOR]

A doxorubicin (DOX)-loaded and light-induced ROS-producing polymeric micelle (D-LRPM), in which light triggers simultaneous DOX-release and endo/lysosomal escape, produces a powerful, spatiotemporally controllable, therapeutic efficacy for tumor treatment. [ABSTRACT FROM AUTHOR]

The article discusses the synthesis of a series of water-insoluble pullulan-g-poly(L-lactide) (PUPL) copolymers with hydrophobic units of different lengths using a one-pot synthetic method and triethylamine (TEA) as a catalyst. It states that the copolymers were dissolved in dimethyl sulfoxide, acetone, and ethanol. It shows that PUPL nanogels may prove useful as anticancer drug carriers because of controlled doxorubicin (DOX) release rate and low critical association concentrations.

In order to construct polymeric nanoparticles responsive to both temperature and pH, three different copolymers (PNDSP 1, 2 and 3), differing in their composition of a thermosensitive co-telomer of N-isopropylacrylamide and N,N-dimethylacrylamide (MNDT, Mn=4600, clouding temperature; 40 °C) and a pH-sensitive sulfamethoxypyridazine telomer (MSPT, Mn=3600, solubility transition; pH 7.4), were synthesized by radical polymerization. Each methacryloyl group terminated telomer was prepared in the presence of 2,2′-azobisisobutyronitrile and 2-aminoethanethiol as a chain transfer agent, followed by coupling with methacryloyl chloride. After the nanoparticles of the copolymers were prepared by diafiltration against the buffers of two different conditions (pH 7.0 and 35 °C (nanoparticle type I); pH 8.0 and 45 °C (nanoparticle type II)), their physicochemical properties in aqueous solutions were characterized by dynamic light scattering (DLS) and fluorescence spectroscopy. The diafiltration condition affected the physicochemical properties of the nanoparticles. The mean diameter of type I nanoparticles was <40 nm, while the diameter of type II ranged from 90 to 140 nm. The critical aggregation concentration (CAC) of type I nanoparticles(>1.2 mg/l) was higher than that of type II (<0.9 mg/l). The type II nanoparticle showed an increased loading efficiency of doxorubicin (DOX), a model anticancer drug. The DOX release rates were influenced by four combinatorial release conditions of two pH levels (7.0 and 7.5) and temperatures (37 and 42 °C). By converting the property of two graft chains (MNDT and MSPT) from hydrophilic (or phobic) to hydrophobic (or philic) simultaneously, the highest DOX release rate from both types of nanoparticles was achieved. The release condition of switching from hydrophilic to hydrophobic surface corresponded to local low tumor pH and hyperthermic conditions. The results indicate that the different types of nanoparticles responsive to local tumor pH, hyperthermic condition, or both can be prepared from the same polymer structure and composition depending on nanoparticle forming condition. [Copyright &y& Elsevier]

Tumor priming is considered a promising strategy for improving drug distribution in malignant tissues. Multicellular layers (MCLs) of human cancer cells are potentially useful models for evaluating tumor-priming agents. We evaluated the priming effects of paclitaxel (PTX) on doxorubicin (DOX) penetration using MCLs of the human colorectal cancer cell lines including DLD-1, HCT-116, and HT-29. The penetration of DOX treated at 50 μM for 3 h was highly limited in all three MCLs. The penetration of the priming agent PTX into MCLs was determined using rhodamine-labeled PTX and appeared to be cell line-dependent: full penetration was observed in HCT-116 and HT-29 MCLs, whereas only limited penetration occurred in DLD-1 MCLs. PTX pretreatment at 20 μM for 24 or 48 h induced a tumor-priming effect in DOX distribution, with a 3 to 5.6-fold-increase in HCT-116 and HT-29 MCLs but a less than two-fold increase in DLD-1 MCLs. PTX treatment decreased fibronectin expression in HCT-116 and HT-29 MCLs but not in DLD-1, suggesting that the prominent priming effect of PTX in HCT-116 and HT-29 MCLs may be associated with the downregulation of fibronectin expression. Our study demonstrated that MCLs of human cancer cells are a useful model not only for the study of drug penetration into tumor tissues but also for screening and evaluating tumor-priming agents. • DOX penetration was highly limited in three colorectal cancer cell line MCLs. • PTX pretreatment significantly enhanced the penetration of DOX into the MCLs. • The PTX priming effect was more pronounced in HCT-116 and HT-29 MCLs. • The high fibronectin levels in HCT-116 and HT-29 MCLs were reduced by PTX priming. • Human cancer cell MCLs are useful for studying drug penetration and tumor-priming. [ABSTRACT FROM AUTHOR]

Abstract: In order to obtain feasibility data regarding the possibility of using chondroitin sulfate (CS) in an anti-cancer drug delivery system, CS was chemically modified by a one-step process with acetic anhydride. Although 3 samples with different degrees of acetylation were synthesized, only the sample with the highest degree of acetylation (AC-CS3) was tested as a nanogel because the others (AC-CS1 and 2) dissolved in distilled water (DW) in the test range (1 - 10mg/ml). The AC-CS3 nanogel was characterized by fluorescence probe and dynamic light scattering (DLS) techniques. Its critical aggregation concentration (CAC) was<2.0×10−2 mg/ml at 25°C. The partition equilibrium constant, Kv, of the nanogel (7.88×105) was similar to that of polymeric micelles, which means that the acetyl group may act as a hydrophobic core controlling pharmacokinetic behavior. The higher surface charge value in the nanogel, above - 40 due to carboxyl and sulfate groups in CS, explains its good stability. The anticancer drug doxorubicin (DOX) loading efficiency of the AC-CS3 nanogel was also superior, at above 90%. Changes in the size of the polydispersion index (PDI) of nanogels loaded with DOX over a 3-week period were negligible. The nanogels interacted with HeLa cells and were internalized together with the entrapped drug within the cytoplasm, probably via an endocytic mechanism exploited by sugar receptors. Based on these results, the AC-CS3 nanogel is expected to prove useful as an anti-cancer drug carrier for chemotherapy. [Copyright &y& Elsevier]

Abstract: Thermo-responsive nanogels from poly(l-lactide)-g-pullulan (PLP1 and 2) copolymers with different lactide contents were investigated as an anticancer drug delivery carrier. The phase transition temperature of PLP 1 with lower lactide content in distilled water showed around 35°C. Upon adding 0.15M NaCl to PLP 1, a significant difference in the transmittance was observed when comparing the non-additive salt condition. The total amount of released doxorubicin (DOX) from the DOX-loaded PLP nanogels increased with increasing temperature for 50h. A noticeable difference in the initial release by PLP 1 was observed between 37 and 42°C. In the 50% inhibitory concentration (IC50) analysis, the IC50 values of DOX released from PLP 1 were approximately 5.9 and 9.3μg/mL at 37 and 42°C, respectively. The results suggest that self-assembled PLP nanogels, by means of a triggering temperature, can be used as a long-term drug delivery system in cancer treatments. [Copyright &y& Elsevier]

Abstract: In order to develop a novel self-assembly as a means of cancer cell targeting, self-organized nanogels were prepared from acetylated hyaluronic acid with low molecular weight (AC-HALM). Three samples were obtained (AC-HALM1, 2 and 3) with degrees of acetylation, 0.8, 2.1, or 2.6 acetyl groups per unit (2 glucose rings) of HALM to control their hydrophobicity. The mean diameters of AC-HALM2 and 3 were less than 300nm with unimodal size distribution, while that of AC-HALM1 was above 400nm. The critical aggregation concentrations (CAC) of the nanogels in distilled water were <1×10−1 mg/mL. The doxorubicine (DOX) loading efficiencies and loading contents of AC-HALM increased as the degree of acetylation increased, in particular, the loading efficiency of AC-HALM3 reached above 90%. AC-HALM3 nanogels showed IC50 at 1300ng/mL of the DOX concentration against HeLa cells (with HA-binding receptors) similar to free DOX. For monitoring of specific interaction with a carcinoma cell line (HeLa cells with HA-binding receptors), AC-HALM3 was labeled with FITC and observed with a confocal microscope. HeLa cells were strongly luminated by interactions with fluorescence-labeled AC-HALM3 nanogels; however, this luminance was significantly decreased by competition inhibition of free HA. This result indicates that modified HA maintains the ability to interact with HA-binding receptors. The selective cytotoxicity and interaction of AC-HALM nanogels may help reduce side effects of anti-cancer drugs in clinical use. [Copyright &y& Elsevier]

Abstract: Recent researches to develop nano-carrier systems in anti-cancer drug delivery have focused on more complicated design to improve therapeutic efficacy and to reduce side effects. Although such efforts have great impact to biomedical science and engineering, the complexity has been a huddle because of clinical and economic problems. In order to overcome the problems, a simplest strategy to fabricate nano-carriers to deliver doxorubicin (DOX) was proposed in the present study. Two significant subjects (i) formation of nanoparticles loading and releasing DOX and (ii) binding specificity of them to cells, were examined. Folic acid (FA) was directly coupled with pullulan (Pul) backbone by ester linkage (FA/Pul conjugate) and the degree of substitution (DS) was varied, which were confirmed by 1H NMR and UV spectrophotometry. Light scattering results revealed that the nanogels possessed two major size distributions around 70 and 270nm in an aqueous solution. Their critical aggregation concentrations (CACs) were less than 10μg/mL, which are lower than general critical micelle concentrations (CMCs) of low-molecular-weight surfactants. Transmission electron microscopy (TEM) images showed well-dispersed nanogel morphology in a dried state. Depending on the DS, the nanogels showed different DOX-loading and releasing profiles. The DOX release rate from FA8/Pul (with the highest DS) for 24h was slower than that from FA4/or FA6/Pul, indicating that the FA worked as a hydrophobic moiety for drug holding. Cellular uptake of the nanogels (KB cells) was also monitored by confocal microscopy. All nanogels were internalized regardless of the DS of FA. Based on the results, the objectives of this study, to suggest a new method overcoming the complications in the drug carrier design, were successfully verified. [Copyright &y& Elsevier]