The Effect of Molecular Structure on Cytotoxicity and Antitumor Activity of PEGylated Nanomedicines.

Fundamental studies on the cellular uptake and drug release of PEGylated nanomedicines are beneficial to understand their fate in vivo and construct ideal nanoparticle formulations. In this work, the detailed metabolic process of PEGylated doxorubicin (Dox) nanomedicines were investigated via confocal laser scanning microscopy (CLSM), flow cytometry (FCM), cytotoxicity test, fluorescence imaging in vivo (FLIV) and liquid chromatography tandem mass spectrometry (LC-MS/MS). Among them, only LC-MS/MS could accurately determine the content of PEGylated Dox and Dox in vitro and in vivo. To the best of our knowledge, this was the first time the PEGylated Dox and released Dox were simultaneously quantified. The interplay of molecular structures, cellular uptake, drug release, and antitumor effect was well characterized. PEG with high molecular weight impeded the cellular uptake of nanoparticles, and the acid-labile hydrazone bond between Dox and PEG promoted Dox release significantly. Cellular uptake and drug release play decisive roles in cytotoxicity and antitumor effect, as evidenced by LC-MS/MS. We emphasized that LC-MS/MS would be a practicable method to quantify PEGylated drugs without complex tags, which could be more in-depth to understand the interaction between PEGylated nanomedicines and their antitumor efficacy.

Stimuli-Responsive Polymersomes for Biomedical Applications.

Polymersomes, the structural analogues of liposomes, are hollow structures enclosed by a bilayer membrane made from amphiphilic copolymers. Polymersomes have been proposed to mimic the structure and properties of cellular membranes and viral capsids. Excellent robustness and stability, chemical versatility for tunable membrane properties and surface functionalization make polymersomes attractive candidates for drug delivery, diagnostic imaging, nanoreactor vessels, and artificial organelles. In further biomimetic strategies, stimuli-responsive polymersomes that can recognize various external physical or internal biological environmental stimuli and conduct "on demand" release in dose-, spatial-, and temporal-controlled fashions have been widely developed. This Perspective focuses on recent advances in stimuli-responsive polymersomes and their potential biomedical applications. Representative examples of each stimulus, the advantages and limitations of different strategies, and the future opportunities and challenges are discussed.

Turning Ineffective Transplatin into a Highly Potent Anticancer Drug via a Prodrug Strategy for Drug Delivery and Inhibiting Cisplatin Drug Resistance.

Clinically ineffective transplatin is highly potent against cancer cells when transformed into a transplatin(IV) prodrug nanoparticle. Herein, a hydrophobic transplatin(IV) was synthesized by H2O2-oxidization of transplatin and attachment of two hydrophobic aliphatic chains. Transplatin(IV) was subsequently encapsulated by a biodegradable amphiphilic copolymer, MPEG-PLA, forming a well-defined spherical micelles (M(TransPt)). Transplatin(IV) was protected efficiently and could be released under a simulated cancerous intracellular condition. Compared to the cisplatin and transplatin, M(TransPt) showed the highest Pt uptake and a clathrin-dependent endocytosis pathway. Most importantly, M(TransPt) displayed a nanomolar IC50 on A2780 cells and a great potency on cisplatin resistant A2780DDP cell line. Overall, this nanoplatform for delivering trans-geometry platinum(IV) drug exhibits excellent characteristics for enhancing efficacy and overcoming cisplatin drug resistance, and holds a strong promise for clinical use in the near future.

Amphiphilic Polycarbonates from Carborane-Installed Cyclic Carbonates as Potential Agents for Boron Neutron Capture Therapy.

Carboranes with rich boron content have showed significant applications in the field of boron neutron capture therapy. Biodegradable derivatives of carborane-conjugated polymers with well-defined structure and tunable loading of boron atoms are far less explored. Herein, a new family of amphiphilic carborane-conjugated polycarbonates was synthesized by ring-opening polymerization of a carborane-installed cyclic carbonate monomer. Catalyzed by TBD from a poly(ethylene glycol) macroinitiator, the polymerization proceeded to relatively high conversions (>65%), with low polydispersity in a certain range of molecular weight. The boron content was readily tuned by the feed ratio of the monomer and initiator. The resultant amphiphilic polycarbonates self-assembled in water into spherical nanoparticles of different sizes depending on the hydrophilic-to-hydrophobic ratio. It was demonstrated that larger nanoparticles (PN150) were more easily subjected to protein adsorption and captured by the liver, and smaller nanoparticles (PN50) were more likely to enter cancer cells and accumulate at the tumor site. PN50 with thermal neutron irradiation exhibited the highest therapeutic efficacy in vivo. The new synthetic method utilizing amphiphilic biodegradable boron-enriched polymers is useful for developing more-selective and -effective boron delivery systems for BNCT.

Nanoparticle-mediated delivery of multinuclear platinum(IV) prodrugs with enhanced drug uptake and the activity of overcoming drug resistance.

Here, a nanoparticle-mediated delivery of multinuclear platinum(IV) prodrugs [biodegradable polymer-di-cisPt(IV)] for overcoming cisplatin drug resistance is reported. From the MTT assays, lower IC50 values of polymer-di-cisPt(IV) on A2780DDP cells than A2780 were observed with the lowest resistance factor of 0.7. Inductively coupled plasma mass spectroscopy results showed that more drugs were delivered into cancer cells and greater number of Pt-DNA adducts were formed with the use of the polymer-di-cisPt(IV) conjugate nanoparticles. By a mechanistic study with endocytosis inhibitors to treat A2780 cells, we proved that polymer-di-cisPt(IV) conjugate nanoparticles were internalized by the cancer cells through endocytosis rather than through passive diffusion or copper transporter 1-mediated active transportation. This well illustrates the way how the polymer-di-cisPt(IV) micelles overcome cisplatin resistance.

Dual-Sensitive Charge-Conversional Polymeric Prodrug for Efficient Codelivery of Demethylcantharidin and Doxorubicin.

A tumor is a complicated system, and tumor cells are typically heterogeneous in many aspects. Polymeric drug delivery nanocarriers sensitive to a single type of biosignals may not release cargos effectively in all tumor cells, leading to low therapeutic efficacy. To address the challenges, here, we demonstrated a pH/reduction dual-sensitive charge-conversional polymeric prodrug strategy for efficient codelivery. Reduction-sensitive disulfide group and acid-labile anticancer drug (demethylcantharidin, DMC)-conjugated ß-carboxylic amide group were repeatedly and regularly introduced into copolymer chain simultaneously via facile CuAAC click polymerization. The obtained multifunctional polymeric prodrug P(DMC), mPEG-b-poly(disulfide-alt-demethylcantharidin)-b-mPEG was further utilized for DOX encapsulation. Under tumor tissue/cell microenvironments (pH 6.5 and 10 mM GSH), the DOX-loaded polymeric prodrug nanoparticles (P(DMC)@DOX NPs) performed surface negative-to-positive charge conversion and accelerated/sufficient release of DMC and DOX. The remarkably enhanced cellular internalization and cytotoxicity in vitro, especially against DOX-resistant SMMC-7721 cells, were demonstrated. P(DMC)@DOX NPs in vivo also exhibited higher tumor accumulation and improved antitumor efficiency compared to P(SA)@DOX NPs with one drug and without charge-conversion ability. The desired multifunctional polymeric prodrug strategy brings a new opportunity for cancer chemotherapy.

Doxorubicin-Loaded Carborane-Conjugated Polymeric Nanoparticles as Delivery System for Combination Cancer Therapy.

Carborane-conjugated amphiphilic copolymer nanoparticles were designed to deliver anticancer drugs for the combination of chemotherapy and boron neutron capture therapy (BNCT). Poly(ethylene glycol)-b-poly(L-lactide-co-2-methyl-2(2-dicarba-closo-dodecarborane)propyloxycarbonyl-propyne carbonate) (PLMB) was synthesized via the versatile reaction between decaborane and side alkynyl groups, and self-assembled with doxorubicin (DOX) to form drug-loaded nanoparticles. These DOX@PLMB nanoparticles could not only suppress the leakage of the boron compounds into the bloodstream due to the covalent bonds between carborane and polymer main chains, but also protect DOX from initial burst release at physiological conditions because of the dihydrogen bonds between DOX and carborane. It was demonstrated that DOX@PLMB nanoparticles could selectively deliver boron atoms and DOX to the tumor site simultaneously in vivo. Under the combination of chemotherapy and BNCT, the highest tumor suppression efficiency without reduction of body weight was achieved. This polymeric nanoparticles delivery system could be very useful in future chemoradiotherapy to obtain improved therapeutic effect with reduced systemic toxicity.

Use of asymmetric multilayer polylactide nanofiber mats in controlled release of drugs and prevention of liver cancer recurrence after surgery in mice.

Local tumor recurrence remains a major clinical problem following surgical treatment for most cancers such as hepatocellular carcinoma (HCC). An implantable local drug delivery system may be suitable for addressing this unmet clinical need. In this study, asymmetric multilayer polylactide nanofiber (AMPN) mats were prepared and a one-sided and prolonged release profile of hydrophilic dye or oxaliplatin was observed after they were sandwiched between two liver lobes in mice. Covering the surgery site by drug-loaded AMPN mat after tumor resection, in both subcutaneous and orthotopic HCC model in mice, the recurrence of HCC was significantly retarded and the survival time of mice was markedly prolonged. In conclusion, post-surgical therapy at tumor resection margins by drug-loaded AMPN mats may represent a suitable application of nanofiber-based local chemotherapy. FROM THE CLINICAL EDITOR: After cancer surgery, local recurrence remains a significant problem. In this study, the authors designed asymmetric multilayer PLA nanofiber (AMPN) mats and loaded them with anti-tumor drugs. Both in-vitro and in-vivo experiments showed good efficacy in preventing tumor recurrence. This novel product may point a way to the future and improve survival of cancer patients.

A reduction-sensitive carrier system using mesoporous silica nanospheres with biodegradable polyester as caps.

Mesoporous silica nanoparticles (MSN)-polymer hybrid combined with the aliphatic biodegradable polyester caps on the surface were first developed in order to manipulate the smart intracellular release of anticancer drugs. First, poly(ethylene glycol)-b-poly(ε-caprolactone) (PEG-PCL) was successfully grafted on the surface of MSN via disulfide bonds which could cleave under a reduction environment in tumor cells. The anticancer drug doxorubicin (DOX) was encapsulated into the particle pores. The in vitro drug release profile showed that DOX release was significantly restricted by the polymer caps at pH 7.4, while it was greatly accelerated upon the addition of GSH. Cytotoxicity evaluation showed good biocompatibility with the hybrid particles. Fast endocytosis and intracellular DOX release were observed by confocal laser scanning microscopy (CLSM). The DOX-loaded particles exhibited comparable antitumor activity with free DOX towards HeLa cells and showed in a time-dependent manner. This work developed an extensive method of utilizing aliphatic biodegradable polyesters as polymer caps for MSN to control drug delivery. The paper might offer a potential option for cancer therapy.

Biological characterization of folate-decorated biodegradable polymer-platinum(II) complex micelles.

A biodegradable and amphiphilic copolymer, poly(ethylene glycol)-block-poly(l-lactide-co-2-methyl-2-carboxyl-propylene carbonate) (mPEG-b-P(LA-co-MCC)), which contains pendant carboxyl groups, was chosen as a drug carrier for the active anticancer part (diaminocyclohexane platinum, DACH-Pt) of oxaliplatin to form mPEG-b-P(LA-co-MCC/Pt) complex. A folic acid-conjugated copolymer, folic acid-poly(ethylene glycol)-block-poly(L-lactide) (FA-PEG-PLA), with similar chemical structure was chosen for targeting. Multifunctional micelles were successfully prepared by a coassembling method. In vitro evaluation was performed by using SKOV-3 and MCF-7 cancer cells. In vivo blood clearance of platinum was studied, and the results show that micelles exhibit longer blood circulation after iv injection. Pt biodistribution was studied by measuring its levels in plasma, organs, and tumors, especially in tumor cell DNA, by atomic absorption and inductively coupled plasma mass spectrometry. Antitumor activity was assessed in mice bearing H22 liver cancers, and the results showed that the micelles with FA moieties exhibited greater antitumor efficacy than those without FA or oxaliplatin. Therefore, these novel multifunctional platinum micelles have great potential in future clinical application.

Transferrin-conjugated micelles: enhanced accumulation and antitumor effect for transferrin-receptor-overexpressing cancer models.

As the transport protein for iron, transferrin can trigger cellular endocytosis once binding to its receptor (TfR) on the cell membrane. Using this property, we conjugated transferrin onto the surface of biodegradable polymeric micelles constructed from amphiphilic block copolymers. The core of micelle was either labeled with a near-infrared dye (NIR) or conjugated with a chemotherapeutic drug paclitaxel (PTX) to study the biodistribution or antitumor effect in nude mice bearing subcutaneous TfR-overexpressing cancers. DLS and TEM showed that the sizes of Tf-conjugated and Tf-free micelles were in the range of 85-110 nm. Confocal laser scanning microscopy and flow cytometry experiments indicated that the uptake efficiency of the micelles by the TfR-overexpressing cells was enhanced by Tf conjugation. Semiquantitative analysis of the NIR signals collected from the tumor site showed that the maximum accumulation was achieved at 28 h in the M(NIR) group, while at 22 h in Tf-M(NIR) groups; and the area under the intensity curve in the Tf-M(NIR) groups was more than that in M(NIR) group. Finally, the tumor inhibition effects of targeting micelles were studied with the gastric carcinoma model which overexpressed TfR. The analysis of tumor volumes and the observation of H&E-stained tumor sections showed that Tf-M(PTX) had the best antitumor effect compared with the control groups (saline, PTX, and M(PTX)). The results of this study demonstrated the potential application of Tf-conjugated polymeric micelles in the treatment of TfR-overexpressing cancers.

Biodegradable amphiphilic copolymer containing nucleobase: synthesis, self-assembly in aqueous solutions, and potential use in controlled drug delivery.

Biodegradable nucleobase-grafted amphiphilic copolymer, the methoxyl poly (ethylene glycol)-b-poly (L-lactide-co-2-methyl-2(3-(2,3-dihydroxylpropylthio) propyloxycarbonyl)-propylene carbonate/1-carboxymethylthymine) (mPEG-b- P(LA-co-MPT)), was synthesized. (1)H NMR titration and FT-IR spectroscopy indicated that the hydrogen-bonding could be formed between mPEG-b-P(LA-co-MPT) and 9-hexadecyladenine (A-C16). The hydrophobic microenvironment of the amphiphilic copolymer can protect the complementary multiple hydrogen bonds between mPEG-b-P(LA-co-MPT) and A-C16 from water effectively. The addition of A-C16 not only lowered the critical aggregation concentration (CAC) of mPEG-b-P(LA-co-MPT)/A-C16 nanoparticles (NPs) in aqueous solution but also induced different morphologies, which can be observed by transmission electron microscopy (TEM). Meanwhile, dynamic light scattering (DLS) and turbidometry was utilized to evaluate the effect of temperature and pH change on the stability of mPEG-b-P(LA-co-MPT)/A-C16 NPs. Cytotoxicity evaluation showed good biocompatibility of the mPEG-b-P(LA-co-MPT)/A-C16 NPs. The in vitro drug release profile showed that with the increase of A-C16 content, the doxorubiucin (DOX) release at pH 7.4 decreased, while the faster release rate was observed with the addition of A-C16 with a pH of 5.0. Importantly, DOX-loaded NPs exerted comparable cytotoxicity against MDA-MB-231 cells. This work provided a new method to stabilize NP structure using hydrogen-bonds and would have the potential to be applied in controlled drug delivery.

Hypoxia-Activated PEGylated Paclitaxel Prodrug Nanoparticles for Potentiated Chemotherapy.

Developing controlled drug-release systems is imperative and valuable for increasing the therapeutic index. Herein, we synthesized hypoxia-responsive PEGylated (PEG = poly(ethylene glycol)) paclitaxel prodrugs by utilizing azobenzene (Azo) as a cleavable linker. The as-fabricated prodrugs could self-assemble into stable nanoparticles (PAP NPs) with high drug content ranging from 26 to 44 wt %. The Azo group in PAP NPs could be cleaved at the tumorous hypoxia microenvironment and promoted the release of paclitaxel for exerting cytotoxicity toward cancer cells. In addition, comparative researches revealed that the PAP NPs with the shorter methoxy-PEG chain (molecular weight = 750) possessed enhanced tumor suppression efficacy and alleviated off-target toxicity. Our work demonstrates a promising tactic to develop smart and simple nanomaterials for disease treatment.

Synthesis of OH-group-containing, biodegradable polyurethane and protein fixation on its surface.

A series of biodegradable polyurethanes containing free side hydroxyl groups (PUOH) were synthesized successfully in two steps: (1) PLA diol as soft segment, hexamethylene diisocyanate (HDI) as hard segment, and benzalpentaerythritol (BPO) as a chain extender were used to synthesize PUs with protected OH groups; (2) CF(3)COOH was used as a deprotection agent to remove the benzal groups on PU to prepare PUOH. The properties of PU and PUOH were characterized by Fourier transform infrared spectroscopy (FT-IR), nuclear magnetic resonance (NMR), differential scanning calorimetry (DSC), water contact angle measurement, and gel permeation chromatography (GPC). The benzal groups were removed completely in 15 min without detrimental effect on PU main chains to obtain PUOHs. 4-Azidobenzoic acid was conjugated to PUOH through its esterification with the free OH groups on PUOH. The results of immunofluorescence assay showed that the phenyl azide groups formed were capable of binding mouse IgG under UV (254 nm) irradiation in 3 min; the bound mouse IgG retained its own biological activity and could further bind the FITC-labeled anti(mouse IgG). Therefore, this material has a potential in immunofluorescence assay and related fields.

Chemosynthesis of poly(ε-lysine)-analogous polymers by microwave-assisted click polymerization.

Poly(ε-lysine) (ε-PL)-analogous click polypeptides with not only similar α-amino side groups but also similar main chain to ε-PL were chemically synthesized for the first time through click polymerization from aspartic (or glutamic)-acid-based dialkyne and diazide monomers. With microwave-assisting, the reaction time of click polymerization was compressed into 30 min. The polymers were fully characterized by NMR, ATR-FTIR, and SEC-MALLS analysis. The deprotected click polypeptides had similar pK(a) value (7.5) and relatively low cytotoxicity as ε-PL and could be used as substitutes of ε-PL in biomedical applications, especially in endotoxin selective removal. Poly(ethylene glycol) (PEG)-containing alternating copolymers with α-amino groups were also synthesized and characterized. After deprotection, the polymers could be used as functional gene vector with PEG shadowing system and NCA initiator to get amphiphilic graft polymers.

RGD-conjugated copolymer incorporated into composite of poly(lactide-co-glycotide) and poly(L-lactide)-grafted nanohydroxyapatite for bone tissue engineering.

Various surface modification methods of RGD (Arg-Gly-Asp) peptides on biomaterials have been developed to improve cell adhesion. This study aimed to examine a RGD-conjugated copolymer RGD/MPEG-PLA-PBLG (RGD-copolymer) for its ability to promote bone regeneration by mixing it with the composite of poly(lactide-co-glycotide) (PLGA) and hydroxyapatite nanoparticles surface-grafted with poly(L-lactide) (g-HAP). The porous scaffolds were prepared using solvent casting/particulate leaching method and grafted to repair the rabbit radius defects after seeding with autologous bone marrow mesenchymal cells (MSCs) of rabbits. After incorporation of RGD-copolymer, there were no significant influences on scaffold's porosity and pore size. Nitrogen of RGD peptide, and calcium and phosphor of g-HAP could be exposed on the surface of the scaffold simultaneously. Although the cell viability of its leaching liquid was 92% that was lower than g-HAP/PLGA, its cell adhesion and growth of 3T3 and osteoblasts were promoted significantly. The greatest increment in cell adhesion ratios (131.2-157.1% higher than g-HAP/PLGA) was observed when its contents were 0.1-1 wt % but only at 0.5 h after cell seeding. All the defects repaired with the implants were bridged after 24 weeks postsurgery, but the RGD-copolymer contained composite had larger new bone formation and better fusion interface. The composites containing RGD-copolymer enhanced bone ingrowth but presented more woven bones than others. The combined application of RGD-copolymer and bone morphological protein 2 (BMP-2) exhibited the best bone healing quality and was recommended as an optimal strategy for the use of RGD peptides.

Intracellular Enzyme-Responsive Profluorophore and Prodrug Nanoparticles for Tumor-Specific Imaging and Precise Chemotherapy.

Responsive drug delivery systems possess great potential in disease diagnosis and treatment. Herein, we develop an activatable prodrug and fluorescence imaging material by engineering the endogenous NAD(P)H:quinone oxidoreductase-1 (NQO1) responsive linker. The as-prepared nanomaterials possess the NQO1-switched drug release and fluorescence enablement, which realizes the tumor-specific chemotherapy and imaging in living mice. The enzyme-sensitive prodrug nanoparticles exhibit selectively potent anticancer performance to NQO1-positive cancer and ignorable off-target toxicity. This work provides an alternative strategy for constructing smart prodrug nanoplatforms with precision, selectivity, and practicability for advanced cancer imaging and therapy.

Multi-armed poly(L-glutamic acid)-graft-oligoethylenimine copolymers as efficient nonviral gene delivery vectors.

BACKGROUND: The application of polyethylenimine (PEI) in gene delivery has been severely limited by significant cytotoxicity that results from a nondegradable methylene backbone and high cationic charge density. It is therefore necessary to develop novel biodegradable PEI derivates for low-toxic, highly efficient gene delivery. METHODS: A series of novel cationic copolymers with various charge density were designed and synthesized by grafting different kinds of oligoethylenimine (OEI) onto a determinate multi-armed poly(L-glutamic acid) backbone. The molecular structures of multi-armed poly(L-glutamic acid)-graft-OEI (MP-g-OEI) copolymers were characterized using nuclear magnetic resonance, viscosimetry and gel permeation chromatography. Moreover, the MP-g-OEI/DNA complexes were measured by a gel retardation assay, dynamic light scattering and atomic force microscopy to determine DNA binding ability, particle size, zeta potential, complex formation and shape, respectively. MP-g-OEI copolymers were also evaluated in Chinese hamster ovary and human embryonic kidney-293 cells for their cytotoxicity and transfection efficiency. RESULTS: The particle sizes of MP-g-OEI/DNA complexes were in a range of 109.6-182.6 nm and the zeta potentials were in a range of 29.2-44.5 mV above the N/P ratio of 5. All the MP-g-OEI copolymers exhibited lower cytotoxicity and higher gene transfection efficiency than PEI25k in the absence and presence of serum with different cell lines. Importantly, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay revealed that the cytotoxicity of MP-g-OEI copolymers varied with their molecular weight and charge density, and two of MP-g-OEI copolymers (OEI600-MP and OEI1800-MP) could achieve optimal transfection efficiency at a similar low N/P ratio as that for PEI25k. CONCLUSIONS: MP-g-OEI copolymers demonstrated considerable potential as nonviral vectors for gene therapy.

Synthesis and characterization of multifunctional poly(glycidyl methacrylate) microspheres and their use in cell separation.

Multifunctional poly(glycidyl methacrylate) (PGMA) microspheres containing magnetic, fluorescent, and cancer cell-specific moieties were prepared in four steps: (i) preparation of parent PGMA microspheres by dispersion polymerization and their reaction with ethylenediamine to obtain amino groups, (ii) precipitation of iron ions (Fe(2+) and Fe(3+)) to form Fe(3)O(4) nanoparticles within the microspheres, (iii) consecutive reactions of folic acid with the amino groups on PGMA, and (iv) incorporation of fluorescein isothiocyanate into the microspheres. The microspheres were superparamagnetic, highly monodispersive, intensively fluorescent, and capable of recognizing and binding cancer cells that overexpress folic acid receptors. It was demonstrated that with these microspheres, HeLa cells could be captured from their suspension and easily moved in the direction of the externally applied magnetic field.

Biodegradable block copolymer-doxorubicin conjugates via different linkages: preparation, characterization, and in vitro evaluation.

Doxorubicin (Dox) was conjugated onto a biodegradable block copolymer methoxy-poly(ethylene glycol)-block-poly(lactide-co-2,2-dihydroxymethylpropylene carbonate (mPEG-b-P(LA-co-DHP)) via a carbamate linkage and an acid-labile hydrazone linkage, respectively. Mutifunctional mixed micelles consisting of Dox-containing copolymer mPEG-b-P(LA-co-DHP/Dox) and folic acid-containing copolymer mPEG-b-P(LA-co-DHP/FA) were successfully prepared by coassembling the two component copolymers. The mixed micelles had well-defined core shell structure and their diameters were in the range of 70-100 nm. Both Dox-conjugates (via carbamate or hydrazone linkage) showed pH-dependent release behavior, and the micelles with hydrazone linkage showed more pH-sensitivity compared to those with carbamate linkage. The in vitro cell uptake experiment by CLSM and flow cytometry showed preferential internalization of FA-containing micelles by human ovarian cancer cell line SKOV-3 than that without FA. Flow cytometric analysis was conducted to reveal the enhanced cell apoptosis caused by the FA-containing micelles. These results suggested that these micelles containing both chemotherapeutic and targeting ligand could be a promising nanocarrier for targeting the drugs to cancer cells and releasing the drug molecules inside the cancer cells.