Enhanced Anti-Tumor Effect of Folate-Targeted FA-AMA-hyd-DOX Conjugate in a Xenograft Model of Human Breast Cancer.

Folate-aminocaproic acid-doxorubicin (FA-AMA-hyd-DOX) was firstly synthesized by our group. It was indicated that FA-AMA-hyd-DOX was pH-responsive, and had strong cytotoxicity on a folate receptor overexpressing cell line (KB cells) in vitro. The aim of our study was to further explore the potential use of FA-AMA-hyd-DOX as a new therapeutic drug for breast cancer. The cellular uptake and the antiproliferative activity of the FA-AMA-hyd-DOX in MDA-MB-231 cells were measured. Compared with DOX, FA-AMA-hyd-DOX exhibited higher targeting ability and cytotoxicity to FR-positive tumor cells. Subsequently, the tissue distribution of FA-AMA-hyd-DOX was studied, and the result confirmed that DOX modified by FA can effectively increase the selectivity of drugs in vivo. After determining the maximum tolerated dose (MTD) of FA-AMA-hyd-DOX in MDA-MB-231 tumor-bearing nude mice, the antitumor effects and the in vivo safety of FA-AMA-hyd-DOX were systematically evaluated. The data showed that FA-AMA-hyd-DOX could effectively increase the dose of DOX tolerated by tumor-bearing nude mice and significantly inhibit MDA-MB-231 tumor growth in vivo. Furthermore, FA-AMA-hyd-DOX treatment resulted in almost no obvious damage to the mice. All the positive data suggest that FA-targeted FA-AMA-hyd-DOX is a promising tumor-targeted compound for breast cancer therapy.

Novel Synthetic Strategies Enable the Efficient Development of Folate Conjugates for Cancer Radiotheranostics.

The folate receptor (FR) is an interesting target for radiotheranostics due to its overexpression in several tumor types. The progress in developing novel folate radioconjugates is, however, slow due to the synthetic challenges that folate chemistry presents. The goal of this study was, thus, to establish versatile solid-phase synthetic strategies for a convenient preparation of novel folate conjugates. Two approaches were established based on an orthogonal fluorenylmethyloxycarbonyl (Fmoc)-protection strategy to enable a modular buildup of an albumin-binding DOTA conjugate (known as OxFol-1) using folic acid (oxidized folate version) as a targeting agent. The main difference between the two approaches was the sequence of conjugating the single structural units. The approach that introduced the folate entity as the last unit appeared particularly useful for the preparation of conjugates based on 6R- or 6S-5-methyltetrahydrofolic acid (5-MTHF; a reduced folate version) as targeting entity. Three types of folate conjugates were synthesized either with a p-iodophenyl-based albumin binder (OxFol-1, 6R-RedFol-1, and 6S-RedFol-1) or without an albumin-binding entity (OxFol-14, 6R-RedFol-14, and 6S-RedFol-14). All six conjugates were obtained with high chemical purity (>98%) after 9-13 synthesis steps and a single final HPLC purification. Radiolabeling with lutetium-177 was feasible at high molar activity, and the resulting radioconjugates were stable over at least 24 h. Biodistribution and SPECT/CT imaging studies confirmed the favorable effect of an albumin-binding entity to increase the tumor uptake and reduce kidney retention of folate radioconjugates. The increased tumor-to-kidney ratios obtained with [177Lu]Lu-6R-RedFol-1 and [177Lu]Lu-6S-RedFol-1 as compared to [177Lu]Lu-OxFol-1 indicated that 5-MTHF is the preferred FR-targeting agent for albumin-binding radioconjugates. This was, however, not the case for folate radioconjugates without an albumin binder. Thanks to the established synthesis strategy, the preparation of further folate radioconjugates will be facilitated, potentially enabling the optimization of the tissue distribution characteristics even more.

Efficient Design to Monitor the Site-specific Sustained Release of a Non-Emissive Anticancer Drug.

A pH-responsive smart nanocarrier with significant components was synthesized by conjugating the non-emissive anticancer drug methyl orange and polyethylene glycol derived folate moiety to the backbone of polynorbornene. Complete synthesis procedure and characterization methods of three monomers included in the work: norbornene-derived Chlorambucil (Monomer 1), norbornene grafted with polyethylene glycol, and folic acid (Monomer 2) and norbornene attached methyl orange (Monomer 3) connected to the norbornene backbone through ester linkage were clearly discussed. Finally, the random copolymer CHO PEG FOL METH was synthesized by ring-opening metathesis polymerization (ROMP) using Grubbs' second-generation catalyst. Advanced polymer chromatography (APC) was used to find the final polymer's molecular weight and polydispersity index (PDI). Dynamic light scattering, scanning electron microscopy (SEM), and transmission electron microscopy (TEM) were utilized to explore the prodrug's size and morphology. Release experiments of the anticancer drug, Chlorambucil and the coloring agent, methyl orange, were performed at different pH and time. Cell viability assay was carried out for determining the rate of survived cells, followed by the treatment of our final polymer named CHO PEG FOL METH.

A multiple environment-sensitive prodrug nanomicelle strategy based on chitosan graftomer for enhanced tumor therapy of gambogic acid.

A novel multiple environment-sensitive polymeric prodrug of gambogic acid (GA) based on chitosan graftomer was fabricated for cancer treatment. Folic acid-chitosan conjugates was complexed with thermosensitive amine terminated poly-N-isopropylacrylamide (NH2-PNIPAM) to develop FA-CSPN. Gambogic acid was conjugated with the graftomer via esterification to achieve high drug-loading capacity and controlled drug release. The resulting amphiphilic prodrug, O-(gambogic acid)-N-(folic acid)-N'-(NH2-PNIPAM) chitosan graftomer (GFCP), could self-assemble into micelles. As expected, the micelles were stable and biocompatible, featuring pH-, esterase- and temperature-dependent manner of drug release. Moreover, the anticancer effect studies of GFCP micelles were performed using a tumor-bearing mouse model and cellular assays (tumor cell uptake assay, cytotoxicity and tumor-sphere penetration). Collectively, GFCP micelles show both potential in vivo and in vitro in improving the anticancer effectiveness of GA owing to high loading capacity, targeted tumor accumulation, and multiple tumor microenvironmental responsiveness.

Synthesis of Some New Folic Acid-Based Heterocycles of Anticipated Biological Activity.

To date, no fused heterocycles have been formed on folic acid molecules; for this reason, and others, our target is to synthesize new derivatives of folic acid as isolated or fused systems. Folic acid 1 reacted with ethyl pyruvate, triethyl orthoformate, ethyl chloroformate, thioformic acid hydrazide, and aldehydes to give new derivatives of folic acid 2-6a,b. Moreover, It reacted with benzylidene malononitrile, acetylacetone, ninhydrin, ethyl acetoacetate, ethyl cyanoacetate, and ethyl chloroacetate to give the pteridine fused systems 10-15, respectively. Ethoxycarbonylamino derivate 5 reacted with some nucleophiles containing the NH2 group, such as aminoguanidinium hydrocarbonate, hydrazine hydrate, glycine, thioformic acid hydrazide, and sulfa drugs in different conditions to give the urea derivatives 16-20a,b. Compound 4 reacted with the same nucleophiles to give the methylidene amino derivatives 21-24a,b. The fused compound 10 reacted with thioglycolic acid carbon disulfide, malononitrile, and formamide to give the four cyclic fused systems 25-30, respectively. The biological activity of some synthesized showed moderate effect against bacteria, but no effect shown towards fungi.

Vitamin B9 derivatives as carriers of bioactive cations for musculoskeletal regeneration applications: Synthesis, characterization and biological evaluation.

The development of new drugs for musculoskeletal regeneration purposes has attracted much attention in the last decades. In this work, we present three novel vitamin B9 (folic acid)-derivatives bearing divalent cations (ZnFO, MgFO and MnFO), providing their synthesis mechanism and physicochemical characterization. In addition, a strong emphasis has been placed on evaluating their biological properties (along with our previously reported SrFO) using human mesenchymal stem cells (hMSC). In all the cases, pure folate derivatives (MFOs) with a bidentate coordination mode between the metal and the folate anion, and a 1:1 stoichiometry, were obtained in high yields. A non-cytotoxic dose of all the MFOs (50 µg/mL) was demonstrated to modulate by their own the mRNA profiles towards osteogenic-like or fibrocartilaginous-like phenotypes in basal conditions. Moreover, ZnFO increased the alkaline phosphatase activity in basal conditions, while both ZnFO and MnFO increased the matrix mineralization degree in osteoinductive conditions. Thus, we have demonstrated the bioactivity of these novel compounds and the suitability to further studied them in vivo for musculoskeletal regeneration applications.

Lipoprotein-Inspired Nanocarrier Composed of Folic Acid-Modified Protein and Lipids: Preparation and Evaluation of Tumor-Targeting Effect.

BACKGROUND: Reconstituted lipoproteins (rLips) based on endogenous lipid nanostructures has been increasingly regarded as an excellent and promising antitumor drug delivery. However, some problems relating to the main component, apolipoprotein, for instance, rare source, unaffordable price, and low specificity of relevant receptor expression, become chief obstacles to its broad development and application. PURPOSE: The primary aim of this study is to develop biomimetic rLips by utilizing folic acid (FA)-modified bovine serum albumin (BSA) as a replacement for apolipoprotein and demonstrate its tumor targeting and antitumor efficacy. METHODS: The amino groups of BSA were covalently conjugated with FA through the amide reaction. PTX-loaded nanostructured lipid carrier (termed as P-NLC) consisting of phospholipid, cholesteryl ester, triglyceride and cholesterol was prepared by the emulsification-evaporation method and utilized as the lipid core. FA-modified BSA (FA-BSA) was characterized for the protein substitute degree and attached with NLC by incubation-insert method to form the lipoprotein-mimic nanocomplex (termed as PFB-rLips). The morphology of nanoparticles was observed under transmission electron microscopy (TEM), and the particle size and zeta potential were determined using dynamic light scattering. In vitro release behavior of PTX from PFB-rLips was investigated with the dialysis method. Hemolysis tests were conducted to evaluate the biosecurity of PFB-rLips. Cell uptake and cytotoxicity assays were performed on human hepatocytes (LO2) and human hepatoma cells (HepG2). Tumor targeting was assessed using in vivo imaging system in H22 tumor-bearing mice model. Antitumor efficacy in vivo was investigated and compared between Taxol® (paclitaxel) formulation and PTX-incorporated nanoparticles in the same tumor model. RESULTS: A fixed molar ratio 50:1 of FA to BSA was chosen as the optimal input ratio based on the balance between appropriate degree of protein substitution and amphiphilicity of FA-BSA. The morphology of FB-rLips exhibited as a homogeneous spherical structure featured by lipid cores surrounded with a cloudy protein shell observed under TEM. The particle size, zeta potential and encapsulation efficiency were 174.6±3.2 nm, -17.26±0.9 mV and 82.2±2.4%, respectively. In vitro release behavior of PTX from PFB-rLips was slow and sustained. The uptake of FB-rLips was much higher in HepG2 cells than in LO2 cells. Furthermore, the uptake of FB-rLips was significantly higher than that of rLips without FA involved (termed as B-rLips) and NLC in HepG2 cells. Hemolysis and cytotoxicity assays showed good biocompatibility of FB-rLips. The internalization mechanism of FB-rLips mainly depended on clathrin-mediated and caveolin-mediated endocytosis coupling with energy consumption, and FA receptors expressed on tumor cells played a critical role in cellular uptake process. CCK-8 studies demonstrated that PFB-rLips exhibited significantly better tumor killing ability than Taxol® (paclitaxel) formulation in vitro. Moreover, FB-rLips produced more excellent tumor-targeting properties than NLC through in vivo imaging assays. On the basis of this, PTX-loaded FB-rLips also performed more remarkable anticancer activity than other therapy groups in H22 tumor-bearing mice. CONCLUSION: FB-rLips would serve as a potential nanocarrier for improving tumor-targeting and therapeutic efficacy while reducing the side effects on normal tissues and organs.

Pharmacokinetic Properties of 68Ga-labelled Folic Acid Conjugates: Improvement Using HEHE Tag.

The folate receptor (FR) is a promising cell membrane-associated target for molecular imaging and radionuclide therapy of cancer (FR-α) and potentially also inflammatory diseases (FR-ß) through use of folic acid-based radioconjugate. FR is often overexpressed by cells of epithelial tumors, including tumors of ovary, cervix, endometrium, lungs, kidneys, etc. In healthy tissues, FR can be found in small numbers by the epithelial cells, mainly in the kidneys. Extremely high undesired accumulation of the folate radioconjugates in the renal tissue is a main drawback of FR-targeting concept. In the course of this work, we aimed to reduce the undesirable accumulation of folate radioconjugates in the kidneys by introducing a histidine/glutamic acid tag into their structure. Two folic acid based compounds were synthesized: NODAGA-1,4-butanediamine-folic acid (FA-I, as control) and NODAGA-[Lys-(HE)2]-folic acid (FA-II) which contains a (His-Glu)2 fragment. In vitro studies with FR (+) cells (KB and others) showed that both compounds have specificity for FR. Introduction of (HE)2-tag does not affect FR binding ability of the conjugates. In vivo biodistribution studies with normal laboratory animals, as well as with KB tumor bearing animals, were carried out. The results showed that introduction of the (HE)2 tag into the structure of folate radioconjugates can significantly reduce the accumulation of these compounds in non-target tissues and important organs (the accumulation in the kidneys is reduced 2-4 times), leaving the accumulation in tumor at least at the same level, and even increasing it.

Study on the optical and biological properties in vitro of IR808-PEG-FA.

IR808, an IR780 derivative, is capable of fluorescently imaging and photodynamic therapy in vitro and in vivo. However, its application is greatly hampered by hydrophobicity, toxicity and nonspecific delivery to the targeting tissue and that causes accumulation in the liver and kidney. In order to overcome these limitations, we prepared IR808-PEG-FA from IR808, amino-terminated poly(ethylene glycol) (NH2 -PEG-NH2 , denoted as PEG) and folate (FA). PEG, an accepted hydrophilic medicinal agent, was introduced to improve hydrophobicity, and FA was used to increase targeting ability of the conjugate. The obtained product provides a good water solubility and stronger light intensity in near infrared (NIR)-imaging, and CCK-8 test demonstrated which had no appreciable toxicity. In addition, the cell uptake results indicated that IR808-PEG-FA was specifically targeted to positive tumors cells with folate receptor (FR) compared with IR808, and thus it may be used as a novel diagnostic agent or imaging-guided agent for cancer treatment. So this article provides a way to improve hydrophobicity, optical stability and targeting ability in the field of nano-probe for fluorochromes.

Dual-ligand functionalized carbon nanodots as green fluorescent nanosensors for cellular dual receptor-mediated targeted imaging.

The conjugation of ligands to nanoparticles as drug delivery systems that target specific cells is a promising approach for the delivery of therapeutic agents to tumor cells. Herein, we prepared green-emission fluorescent carbon nanodots (CNDs) by a facile hydrothermal method with d-(+)-glucosamine hydrochloride and l-aspartic acid as the precursors, then covalently conjugated with folate (FA), polyethyleneimine (PEI) and hyaluronic acid (HA) to develop dual ligand-decorated nanocarriers (FA-PEI-HA-CNDs) for the targeted imaging of cancer cells. FA-PEI-HA-CNDs integrated the excellent fluorescence property of CNDs, and can be used for the real-time and noninvasive location tracking of cancer cells. The cellular uptake study demonstrated that FA-PEI-HA-CNDs markedly improved the internalization efficiency in A-549 cells via folate/CD44 receptor-mediated endocytosis in comparison with that of the A549 cells pretreated with excess FA, HA, and FA and HA. Therefore, these dual folate/CD44 receptor-targeted CNDs (FA-PEI-HA-CNDs) show promising potential for cancer detection, drug delivery, and individualized treatment as performance platforms.

Mono- and Polyassociation Processes of Pentavalent Biotinylated PEI Glycopolymers for the Fabrication of Biohybrid Structures with Targeting Properties.

This study describes new mechanistic insights in the sequential polyassociation of streptavidin with biotinylated poly(ethyleneimine) glycopolymers and biotinylated PEGylated folic acid components for the preparation of biohybrid structures (BHS) for controlled targeting experiments. Characterization of the BHS revealed that during the formation and postfunctionalization of BHS, reversible dissociation and reassociation processes occur. The BHS are stable over weeks after finalizing the equilibrium-driven polyassociation process. Cellular uptake studies showed that this sequential polyassociation involving biotinylated PEGylated folic acid components does not lead to enhanced cellular uptake of the resulting BHS. In contrast, polyplexes, containing small interfering RNA and bioconjugates (1:1 molar ratio between biotinylated glycopolymer and monomeric streptavidin-lectin fusion protein), enabled us to control the targeting of tumor cells as revealed by knockdown of the tumor-associated protein survivin. Overall, this study demonstrates the high potential of (networklike) streptavidin-biotin interactions with a dynamic character in the formation of complex BHS and extracellular matrix materials.

Folate receptor targeted delivery of paclitaxel to breast cancer cells via folic acid conjugated graphene oxide grafted methyl acrylate nanocarrier.

The adaptability, joint with a large surface area, electronic flexibility, high intrinsic mobility, high mechanical strength and supreme thermal conductivity have condensed graphene family materials attractive as technological tools of the drug delivery system. In this present study, investigate a modified graphene oxide-methyl acrylate (GO-g-MA) nanocarrier for targeted anti-cancer drug delivery in breast cancer cells and the GO-g-MA fascinated with folic acidas a targeting ligand to target the cancer cells. Paclitaxel (PTX) was assembled through π-π stacking, hydrophophic interaction on the surface of the GO-g-MA/FA carrier. Structural modification of GO-g-MA, functionalization of targeting ligands GO-g-MA/FA and drug loaded GO-g-MA/FA-PTX was characterized and confirmed through FTIR, XRD, SEM,TEM and AFM analysis. The in-vitro drug release pattern of PTX from the GO-g-MA/FA was examined in different pH ranges. An MTT assay was performed to evaluate the cytotoxicity behaviour of the carrier and PTX loaded nanocarrier in the human breast cancer cell line (MDA-MB-231). GO-g-MA/FA-PTX carrier showed that 39% of cytotoxic effect. Furthermore, the in-vivo (DMBA induced breast cancer rats) studies were carried out and treatment with PTX- loaded GO-g-MA/FA nanocarrier attenuates the levels of mitochondrial citric acids enzymes to near normal.

Evaluation of Novel Tumor-Targeted Near-Infrared Probe for Fluorescence-Guided Surgery of Cancer.

Because the most reliable therapy for cancer involves quantitative resection of all diseased tissue, considerable effort has been devoted to improving a surgeon's ability to locate and remove all malignant lesions. With the aid of improved optical imaging equipment, we and others have focused on developing tumor-targeted fluorescent dyes to selectively illuminate cancer nodules during surgery. We describe here the design, synthesis, optical properties, in vitro and in vivo tumor specificity/affinity, pharmacokinetics, preclinical toxicology, and some clinical application of a folate receptor (FR)-targeted NIR dye (OTL38) that concentrates specifically in cancer tissues and clears rapidly from healthy tissues. We demonstrate that OTL38 binds FR-expressing cells with ∼1 nM affinity and eliminates from receptor negative tissues with a half-time of <30 min. We further show that OTL38 enables visualization of malignant lesions at concentrations less than 100-fold those required to elicit signs of toxicity. Since OTL38 also provides excellent tumor contrast in both murine tumor models and human cancer patients, we conclude that OTL38 constitutes an excellent NIR dye for fluorescence-guided resection of malignant lesions in cancer patients.

Reduction-sensitive CD44 receptor-targeted hyaluronic acid derivative micelles for doxorubicin delivery.

INTRODUCTION: A reduction-sensitive CD44-positive tumor-targetable drug delivery system for doxorubicin (DOX) delivery was developed based on hyaluronic acid (HA)-grafted polymers. MATERIALS AND METHODS: HA was conjugated with folic acid (FA) via a reduction-sensitive disulfide linkage to form an amphiphilic polymer (HA-ss-FA). The chemical structure of HA-ss-FA was analyzed by ultraviolet spectroscopy, Fourier transform infrared spectroscopy, and 1H nuclear magnetic resonance (NMR) spectroscopy. The molecular weight of HA-ss-FA was determined by high-performance gel permeation chromatography. Blank HA-ss-FA micelles and DOX-loaded micelles were prepared and characterized. The reduction responsibility, cellular uptake, and in vivo biodistribution of HA-ss-FA micelles were investigated. RESULTS: DOX-loaded micelles were of high encapsulation efficiency (88.09%), high drug-loading content (22.70%), appropriate mean diameter (100-120 nm), narrow size distribution, and negative zeta potential (-6.7 to -31.5 mV). The DOX release from the micelles was significantly enhanced in reduction environment compared to normal environment. The result of in vitro cytotoxicity assay indicated that the blank micelles were of low toxicity and good biocompatibility and the cell viabilities were >100% with the concentration of HA-ss-FA from 18.75 to 600.00 µg/mL. Cellular uptake and in vivo biodistribution studies showed that DOX-loaded micelles were tumor-targetable and could significantly enhance cellular uptake by CD44 receptor-mediated endocytosis, and the cellular uptake of DOX in CD44-positve A549 cells was 1.6-fold more than that in CD44-negative L02 cells. In vivo biodistribution of HA-ss-FA micelles showed that micelles were of good in vivo tumor targetability and the fluorescence of indocyanine green (ICG)-loaded micelles was 4- to 6.6-fold stronger than free ICG within 6 h in HCCLM3 tumor-bearing nude mice. CONCLUSION: HA-ss-FA is a promising nanocarrier with excellent biocompatibility, tumor targetability, and controlled drug release capability for delivery of chemotherapy drugs in cancer therapy.

Lymphoma-targeted treatment using a folic acid-decorated vincristine-loaded drug delivery system.

PURPOSE: B-cell lymphoma is the most frequently diagnosed lymphoid tumor. Folic acid (FA)-decorated systems were found to be preferentially internalized on the B-cell lymphoma cell line which is reported to express the folate receptor. This study was designed to develop an FA-decorated vincristine (VCR)-loaded system for targeted lymphoma treatment. METHODS: FA-decorated lipid was synthesized. VCR-loaded lipid-polymer hybrid nanoparticles (LPNs) were fabricated. In vitro cell lines and an in vivo lymphoma animal model was used to evaluate the anti B-cell lymphoma effect. RESULTS: FA-decorated, VCR-loaded LPNs (FA-VCR/LPNs) have shown a targeted effect in delivery to B-cell lymphoma cells. FA-VCR/LPNs also showed the highest anti-tumor effect in murine-bearing lymphoma xenografts. CONCLUSION: FA-VCR/LPNs can achieve targeted delivery of VCR, bring about an outstanding therapeutic effect to treat lymphoma, and also reduce the systemic toxicity. FA-VCR/LPNs could be an excellent system for lymphoma therapy.

Synthesis of folate­chitosan nanoparticles loaded with ligustrazine to target folate receptor positive cancer cells.

In addition to its vasodilatory effect, ligustrazine (LZ) improves the sensitivity of multidrug resistant cancer cells to chemotherapeutic agents. To enhance the specificity of LZ delivery to tumor cells/tissues, folate­chitosan nanoparticles (FA­CS­NPs) were synthesized by combination of folate ester with the amine group on chitosan to serve as a delivery vehicle for LZ (FA­CS­LZ­NPs). The structure of folate­chitosan and characteristics of FA­CS­LZ­NPs, including its size, encapsulation efficiency, loading capacity and release rates were analyzed. MCF­7 (folate receptor­positive) and A549 (folate receptor­negative) cells cultured with or without folate were treated with FA­CS­LZ­NPs, CS­LZ­NPs or LZ to determine cancer­targeting specificity of FA­CS­LZ­NPs. Fluorescence intensity of intracellular LZ was observed by laser scanning confocal microscopy, and concentration of intracellular LZ was detected by HPLC. The average size of FA­CS­LZ­NPs was 182.7±0.56 nm, and the encapsulation efficiency and loading capacity was 59.6±0.23 and 15.3±0.16% respectively. The cumulative release rate was about 95% at pH 5.0, which was higher than that at pH 7.4. There was higher intracellular LZ accumulation in MCF­7 than that in A549 cells and intracellular LZ concentration was not high when MCF­7 cells were cultured with folate. These results indicated that the targeting specificity of FA­CS­LZ­NPs was mediated by folate receptor. Therefore, the FA­CS­LZ­NPs may be a potential folate receptor­positive tumor cell targeting drug delivery system that could possibly overcome multidrug resistance during cancer therapy.

Folate receptor-targeted ultrasonic PFOB nanoparticles: Synthesis, characterization and application in tumor-targeted imaging.

In this study, we aimed to determine an effective strategy for the synthesis of folate receptor (FR) targeted-nanoparticles (FRNPs). The nanoparticles used as ultrasound contrast agents (UCAs) were composed of a liquid core of perfluorooctyl bromide (PFOB) liposome and a targeted shell chemically conjugated with folic acid (FA) and polyethylene glycol (PEG). This was done in order to avoid recognition and clearance by the mononuclear phagocyte system [also known as the reticuloendothelial system (RES)] and enhance the targeting capability of the nanoparticles to tumors overexpressing folate receptor (FR). The FRNPs exhibited an average particle size of 301±10.8 nm and surface potential of 39.1±0.43 mV. Subsequently, in vitro, FRNPs labeled with FITC fluorescence dye were visibly uptaken into the cytoplasm of FR-overexpressing cancer cells (Bel7402 and SW620 cells), whereas the A549 cells expressing relatively low levels of FR just bound with few FRNPs. These results demonstrated that FRNPs have a high affinity to FR-overexpressing cancer cells. Additionally, in in vivo experiments, FRNPs achieved a greater enhancement of tumor ultrasound imaging and a longer enhancement time in FR-overexpressing tumors and the Cy7-labeled FRNPs exhibited a relatively high tumor-targeted distribution in FR­overexpressing tumors. Targeted ultrasound and fluorescence imaging revealed that FRNPs have the ability to target FR-overexpressing tumors and ex vivo fluorescence imaging was then used to further verify and confirm the presence of FRNPs in tumor tissues with histological analysis of the tumor slices. On the whole, our data demonstrate that the FRNPs may prove to be a promising candidate for the early diagnosis for FR-overexpressing tumors at the molecular and cellular levels.

Folic acid conjugates with photosensitizers for cancer targeting in photodynamic therapy: Synthesis and photophysical properties.

Recent researches in photodynamic therapy have focused on novel techniques to enhance tumour targeting of anticancer drugs and photosensitizers. Coupling a photosensitizer with folic acid could allow more effective targeting of folate receptors which are over-expressed on the surface of many tumour cells. In this study, different folic acid-OEG-conjugated photosensitizers were synthesized, characterized and their photophysical properties were evaluated. The introduction of an OEG does not significantly improve the hydrophilicity of the FA-porphyrin. All the FA-targeted photosensitizers present good to very good photophysical properties. The best one appears to be Ce6. Molar extinction coefficient, fluorescence and singlet oxygen quantum yields were determined and were compared to the corresponding photosensitizer alone.

In vivo tumor targeting and anti-tumor effects of 5-fluororacil loaded, folic acid targeted quantum dot system.

In this study, we modulated the anti-cancer efficacy of 5-Fluorouracil (5-FU) using a carrier system with enhanced targeting efficacy towards folate receptors (FRs) expressing malignant tissues. The 5-FU drug was loaded onto Mn-ZnS quantum dots (QDs) encapsulated with chitosan (CS) biopolymer and conjugated with folic acid (FA) based on a simple wet chemical method. The formation of 5-FU drug loaded composite was confirmed using Fourier transform infrared spectroscopy (FTIR), thermo gravimetric analysis (TGA) and differential scanning calorimetry (DSC). Furthermore, the in vivo biodistribution and tumor targeting specificity of the 5-FU@FACS-Mn:ZnS in the tumor-bearing mice was conducted based on the Zn(2+) tissue bioaccumulation using inductively coupled plasma (ICP) spectroscopy. In addition to the characterization, the in vitro release profile of 5-FU from the conjugates investigated under diffusion controlled method demonstrated a controlled release behaviour as compared against the release behaviour of free 5-FU drug. The as-synthesized 5-FU@FACS-Mn:ZnS nanoparticle (NP) systemically induced higher level of apoptosis in breast cancer cells in vitro as compared to cells treated with free 5-FU drug following both cell cycle and annexin assays, respectively. Also, the in vivo toxicity assessment of the 5-FU@FACS-Mn:ZnS NPs as compared to the control did not cause any significant increase in the activities of the liver and kidney function biomarkers, malondialdehyde (MDA) and nitric oxide (NO) levels. However, based on the FA-FRs chemistry, the 5-FU@FACS-Mn:ZnS NPs specifically accumulated in the tumor of the tumor-bearing mice and thus contributed to the smaller tumor size and less event of metastasis was observed in the lungs when compared to the tumor-bearing mice groups treated with the free 5-FU drug. In summary, the results demonstrated that the 5-FU@FACS-Mn:ZnS QDs exhibits selective anti-tumor effect in MDA-MB231 breast cancer cells in vitro and 4TI breast cancer cells in vivo, providing a blueprint for improving the 5-FU efficacy and tumor targeting specificity with limited systemic toxicity.

Preparation, characterization and biodistribution in quails of 99mTc-folic acid/chitosan nanostructure.

Chitosan (CS) of low molecular weight is prepared using γ-irradiation method in presence of H2O2 as oxidizing agent. The chemical treatment of folic acid (FA) with low molecular weight CS is carried out to prepare FACS complex based on the reaction between NH2 group of CS and γ-COOH group of FA. The structure and properties of FACS complex was characterized by FT-IR, 1H NMR, UV, SEM, TEM, DLS and XRD analyses. TEM and DLS results showed that FACS complex has nanostructure and the mean size of particles was unimodal with average diameters in the range of 165-252nm. Radiolabeling of FACS complex (99mTc-FACS) was done with Technetium-99m (99mTc). The optimum conditions of labeling were investigated. The labeling yield was 85% at pH=6 and 30min reaction time. The effect of time on the stability of 99mTc-FACS complex was studied and the results revealed that it is stable up to 6h after labeling. Biodistribution studies of 99mTc-FACS complex in Quail showed that it distributed to different organs mainly in blood, liver and kidney. The results revealed that the uptake of 99mTc-FACS in the sexual organs (ovary and ovarian) of female Quail was higher than that in the sexual organs (testes) of male Quail, so 99mTc-FACS could be used to differentiate between them. It also showed that FACS is consumed and more essential in female than that in male.