Cancer Cells Overexpressing Folate Receptors Research Articles

Linear polyethyleneimine-based charge-reversal nanoparticles for nuclear-targeted drug delivery

Cationic polymer polyethyleneimine (PEI) can carry DNA across the cell membrane and enter the nucleus, and thus can be a very useful carrier for nuclear drug delivery; however, its highly positive charges make it toxic and not applicable for systemic drug delivery. Here, well-defined linear PEI (Mn = 1000 or 1500 or 2000)-block-polycaprolactone (Mn = 2000) (LPEI–PCL) was synthesized and used to fabricate a pH-triggered charge-reversal nanoparticle to solve this problem. LPEI's secondary amines are amidized as acid-labile β-carboxylic amides (LPEI/amide–PCL). LPEI/amide–PCL formed negatively charged nanoparticles with very low toxicity and low interaction with cells. Once in an acidic environment, the amides hydrolyze to regenerate the amine groups, producing LPEI–PCL nanoparticles carrying cationic charges. The LPEI–PCL escapes from the lysosomes and traverses into the nucleus. Folic-acid targeting groups are introduced to render the nanoparticles cancer-cell targeting capability. The nanoparticles efficiently enter folate-receptor overexpressing cancer cells and traverse to their nuclei. The DOX loaded in the carrier shows much improved cytotoxicity to cancer cells.

Charge-Reversal Polyamidoamine Dendrimer for Cascade Nuclear Drug Delivery

Polyamidoamine (PAMAM) dendrimers with primary amine termini have been extensively explored as drug and gene carriers owing to their unique properties, but their amine-carried cationic charges cause nonspecific cellular uptakes, systemic toxicity and other severe problems in in vivo applications. In this article, we report a charge-reversal approach that latently deactivates PAMAM's primary amines to negatively charged acid-labile amides in order to inhibit its nonspecific interaction with cells, but regenerates the active PAMAM once in acidic environments. A cascade cancer cell nuclear drug delivery was achieved using the latently amidized PAMAM as the carrier conjugated with folic acid as the targeting group and a DNA-toxin drug camptothecin. The conjugate had low nonspecific interactions with cells, but easily entered cancer cells overexpressing folate receptors via receptor-mediated endocytosis. Subsequently, the endocytosed conjugate was transferred to acidic lysosomes, wherein the active PAMAM carrier was regenerated, escaped from the lysosome and then entered the nucleus for drug release. This reversible deactivation/activation makes PAMAM dendrimers useful nanocarriers for in vivo cancer cell nuclear-targeted drug delivery.

Silica‐Coated Manganese Oxide Nanoparticles as a Platform for Targeted Magnetic Resonance and Fluorescence Imaging of Cancer Cells

AbstractMonodisperse silica‐coated manganese oxide nanoparticles (NPs) with a diameter of ∼35 nm are synthesized and are aminated through silanization. The amine‐functionalized core–shell NPs enable the covalent conjugation of a fluorescent dye, Rhodamine B isothiocyanate (RBITC), and folate (FA) onto their surface. The formed Mn3O4@SiO2(RBITC)–FA core–shell nanocomposites are water‐dispersible, stable, and biocompatible when the Mn concentration is below 50 µg mL−1 as confirmed by a cytotoxicity assay. Relaxivity measurements show that the core–shell NPs have a T1 relaxivity (r1) of 0.50 mM−1 s−1 on the 0.5 T scanner and 0.47 mM−1 s−1 on the 3.0 T scanner, suggesting the possibility of using the particles as a T1 contrast agent. Combined flow cytometry, confocal microscopy, and magnetic resonance imaging studies show that the Mn3O4@SiO2(RBITC)–FA nanocomposites can specifically target cancer cells overexpressing FA receptors (FARs). Findings from this study suggest that the silica‐coated Mn3O4 core–shell NPs could be used as a platform for bimodal imaging (both magnetic resonance and fluorescence) in various biological systems.

Folate-mediated poly(3-hydroxybutyrate-co-3-hydroxyoctanoate) nanoparticles for targeting drug delivery

A novel targeting drug delivery system (TDDS) has been developed. Such a TDDS was prepared by W 1/O/W 2 solvent extraction/evaporation method, adopting poly(3-hydroxybutyrate-co-3-hydroxyoctanoate) [P(HB-HO)] as the drug carrier, folic acid (FA) as the targeting ligand, and doxorubicin (DOX) as the model anticancer drug. The average size, drug loading capacity and encapsulation efficiency of the prepared DOX-loaded, folate-mediated P(HB-HO) nanoparticles (DOX/FA–PEG–P(HB-HO) NPs) were found to be around 240 nm, 29.6% and 83.5%. The in vitro release profile displayed that nearly 50% DOX was released in the first 5 days. The intracellular uptake tests of the nanoparticles (NPs) in vitro showed that the DOX/FA–PEG–P(HB-HO) NPs were more efficiently taken up by HeLa cells compared to non-folate-mediated P(HB-HO) NPs. In addition, DOX/FA–PEG–P(HB-HO) NPs (IC 50 = 0.87 μM) showed greater cytotoxicity to HeLa cells than other treated groups. In vivo anti-tumor activity of the DOX/FA–PEG–P(HB-HO) NPs showed a much better therapeutic efficacy in inhibiting tumor growth, and the final mean tumor volume was 178.91 ± 17.43 mm 3, significantly smaller than normal saline control group (542.58 ± 45.19 mm 3). All these results have illustrated that our techniques for the preparing of DOX/FA–PEG–P(HB-HO) NPs developed in present work are feasible and these NPs are effective in selective delivery of anticancer drug to the folate receptor-overexpressed cancer cells. The new TDDS may be a competent candidate in application in targeting treatment of cancers.

Hydrophobically modified carboxymethyl chitosan nanoparticles targeted delivery of paclitaxel

Specific targeting of tumor cells to achieve higher drug levels in tumor tissue and to overcome the side effects is the major goal in cancer therapy. Nanoparticles encapsulating a hydrophobic core in their nanoreservoir structure were developed as a carrier for a water-insoluble drug, paclitaxel. In the present study, target-oriented nanoparticles based on biodegradable O-carboxymethyl chitosan modified with stearic acid. The surface of the nanoparticles was modified by covalent attachment of folic acid (FA) by simple carbodimide reaction to achieve tumor cell targeting property. Nanoparticles were prepared by the sonication method without involving any surfactants/emulsifiers. The nanoparticles were characterized by various state-of-the-art techniques, including laser light scattering for particles size distribution, field emission scanning electron microscopy and transmission electron microscope for surface morphology. The drug release property and the cytotoxicity of the drug loaded nanoparticles to both cancerous and noncancerous cells were evaluated in cell culture system. To our knowledge, this is the first study demonstrating a FA modified hydrophobically chitosan with paclitaxel-loaded nanoparticles targeting of folate receptor overexpressing cancer cells.

Inorganic Drug‐Delivery Nanovehicle Conjugated with Cancer‐Cell‐Specific Ligand

AbstractThe surface of layered double hydroxide nanoparticles, a potential drug‐delivery nanovehicle, is modified with the cancer‐cell‐specific ligand, folic acid. The surface modification is successfully accomplished through step‐by‐step coupling reactions with aminopropyltriethoxysilane and 1‐ethyl‐3‐(3‐dimethyl aminopropyl)‐carbodiimide. In order to evaluate the cancer‐cell targeting effect of folic‐acid‐grafted layered double hydroxide utilizing fluorescence‐related assay, both layered double hydroxide with and without folic acid moiety are labeled with fluorescein 5′‐isothiocyanate. The uptake of layered double hydroxide and folic acid conjugated into KB and A549 cells is visualized using fluorescence microscopy and measured by flow cytometry. Both chemical and biological assay results demonstrate that the folic acid molecules are indeed conjugated to the surface of layered double hydroxide and thus the selectivity of nanovehicles to cancer cells overexpressing folate receptors increases. In this study, it is suggested that layered double hydroxide nanoparticles can be used as drug‐delivery carriers with a targeting function due to the chemical conjugation with specific ligand.

Synthesis of highly stable folic acid conjugated magnetite nanoparticles for targeting cancercells

A new approach towards the design of folic acid conjugated magnetic nanoparticles forenhancing their site specific intracellular uptake against a folate receptor overexpressingcancer cells is reported. Magnetite nanoparticles were prepared by coprecipitation from anFe3+ and Fe2+ solution followed by surface modification with 2-carboxyethyl phosphonic acid to formcarboxyl group terminated nanoparticles. Then folic acid and fluorescein isothiocyanate(FITC) were conjugated with carboxylic acid functionalized magnetite nanoparticles using2,2′-(ethylenedioxy)-bis-ethylamine. These folate-conjugated nanoparticles were characterizedin terms of their size by dynamic light scattering (DLS) and transmission electronmicroscopy (TEM). Surface functional groups and surface composition were analyzed byFourier transform infrared (FTIR) spectroscopy and x-ray photoelectron spectroscopy(XPS), respectively. Vibration sample magnetometry (VSM) measurements showed thesuperparamagnetic nature of the particles at room temperature. Folate-conjugatedmagnetic nanoparticles are noncytotoxic and receptor mediated internalization by HeLaand B16 melanoma F0 cancer cells was confirmed by flow cytometry and confocalmicroscopy.

Oil-Encapsulating PEO−PPO−PEO/PEG Shell Cross-Linked Nanocapsules for Target-Specific Delivery of Paclitaxel

PEO-PPO-PEO/PEG shell cross-linked nanocapsules encapsulating an oil phase in their nanoreservoir structure was developed as a target-specific carrier for a water-insoluble drug, paclitaxel. Oil-encapsulating PEO-PPO-PEO/PEG composite nanocapsules were synthesized by dissolving an oil (Lipiodol) and an amine-reactive PEO-PPO-PEO derivative in dichloromethane and subsequently dispersing in an aqueous solution containing amine-functionalized six-arm-branched poly(ethylene glycol) by ultrasonication. The resultant shell cross-linked nanocapsules had a unique core/shell architecture with an average size of 110.7 +/- 9.9 nm at 37 degrees C, as determined by dynamic light scattering and transmission electron microscopy. Paclitaxel could be effectively solubilized in the inner Lipiodol phase surrounded by a cross-linked PEO-PPO-PEO/PEG shell layer. The paclitaxel-loaded nanocapsules were further conjugated with folic acid to achieve folate receptor targeted delivery. Confocal microscopy and flow cytometric analysis revealed that folate-mediated targeting significantly enhanced the cellular uptake and apoptotic effect against folate receptor overexpressing cancer cells. The present study suggested that these novel nanomaterials encapsulating an oil reservoir could be potentially applied for cancer cell targeted delivery of various water-insoluble therapeutic and diagnostic agents.

Targeted Photodynamic Therapy Agent with a Built-In Apoptosis Sensor for in Vivo Near-Infrared Imaging of Tumor Apoptosis Triggered by its Photosensitization in Situ

Imaging apoptotic cells or tissues after cancer therapy in situ would be a very useful tool for assessing proper treatment conditions and therapeutic outcome. By combining therapeutic and imaging functions, we have designed a multifunctional, membrane-permeable, and cancer-specific agent that triggers and images apoptosis in targeted cells. We chose photodynamic therapy (PDT) as an appropriate cancer treatment modality and caspase 3 as an apoptosis-specific imaging target. This targeted photodynamic therapy agent with a built-in apoptosis sensor (TaBIAS) induces photodamage only to target cells and simultaneously identifies those that are apoptotic by its near-infrared fluorescence. It contains a fluorescent photosensitizer used as an anticancer drug and a cancer-associated folate receptor homing molecule connected to a caspase 3 cleavable peptide linker that has a fluorescence quencher on the opposing site. We demonstrated that PDT-triggered cleavage of the peptide linker by caspase 3, one of the key executioner caspases, results in a detectable increase in fluorescence in folate receptor-overexpressing cancer cells and tumors. The presence of apoptosis was confirmed in vitro by flow cytometry and ex vivo by Apoptag assay, supporting the ability of TaBIAS to specifically induce and image apoptosis in situ.

Folate Receptor-Mediated Cancer Cell Specific Gene Delivery Using Folic Acid-Conjugated Oligochitosans

Chitosan-mediated gene delivery has gained an increasing interest due to its ability to treat cancers and genetic diseases. However, low transfection efficiency and lack of target specificity limit its application for gene and drug delivery. In the present work, folic acid was covalently conjugated to chitosan as a targeting ligand in an attempt to specifically deliver DNA to folate receptor-overexpressing cancer cells. Folic acid-conjugated chitosan (FACN) was successfully synthesized and characterized by 1H-NMR and is biocompatible. In vitro gene transfer potential of FACN was evaluated in human epithelial ovarian cancer OV2008 cells and human breast cancer MCF-7 cells. FACN at a weight ratio of 10:1 exhibited significantly (< 0.01) enhanced gene transfer potential in folate receptor-overexpressing cancer cells as compared to unmodified chitosan. Transfection of FACN/pDNA nanocomplexes is competitively inhibited by free folic acid, suggesting the specific gene delivery of FACN/pDNA nanocomplexes is achieved through folate receptor-mediated endocytosis. Taken together, these results demonstrate that FACN provides a promising carrier for cancer gene therapy.

Target-Specific Cellular Uptake of PLGA Nanoparticles Coated with Poly(l-lysine)−Poly(ethylene glycol)−Folate Conjugate

Poly(D,L-lactic-co-glycolic acid) (PLGA) nanoparticles with anionic surface charge were surface coated with cationic di-block copolymer, poly(L-lysine)-poly(ethylene glycol)-folate (PLL-PEG-FOL) conjugate, for enhancing their site-specific intracellular delivery against folate receptor overexpressing cancer cells. The PLGA nanoparticles coated with the conjugate were characterized in terms of size, surface charge, and change in surface composition by XPS. By employing the flow cytometry method and confocal image analysis, the extent of cellular uptake was comparatively evaluated under various conditions. PLL-PEG-FOL coated PLGA nanoparticles demonstrated far greater extent of cellular uptake to KB cells, suggesting that they were mainly taken up by folate receptor-mediated endocytosis. The enhanced cellular uptake was also observed even in the presence of serum proteins, possibly due to the densely seeded PEG chains. The PLL-PEG-FOL coated PLGA nanoparticles could be potentially applied for cancer cell targeted delivery of various therapeutic agents.

Pteroyl-γ-glutamate-cysteine synthesis and its application in folate receptor-mediated cancer cell targeting using folate-tethered liposomes

Cell membrane-associated folate receptors are selectively overexpressed in certain human tumors. The high affinity of folic acid for folate receptors provides a unique opportunity to use folic acid as a targeting ligand to deliver chemotherapeutic agents to cancer cells. Folate-tethered liposomes bearing pteroyl-γ-glutamate-cysteine-polyethylene glycol (PEG)-distearoylphosphatidylethanolamine (DSPE) as the targeting component are under investigation as mediators of drug and gene delivery to cancer cells that overexpress folate receptors. Pteroyl-γ-glutamate-cysteine synthesis is one of the crucial starting steps in the preparation of pteroyl-γ-glutamate-cysteine-PEG-DSPE. However, published methods for the synthesis of pteroyl-γ-glutamate-cysteine provide low yields and are not easily reproducible. Therefore, we developed a modified synthetic method for the removal of the N 10-trifluoroacetyl group after cleavage/deprotection that is reliable, is easily reproducible, and has high yield (38%) compared with an unreliable yield of 3–20% with the earlier methods. Folate-tethered liposomes containing calcein or doxorubicin were prepared using pteroyl-γ-glutamate-cysteine-PEG-DSPE as the targeting component along with nontargeted liposomes with PEG-DSPE. The results of the uptake of calcein and cytotoxicity of doxorubicin in human cervical cancer HeLa-IU 1 cells and human colon cancer Caco-2 cells demonstrated that folate-tethered liposomes were efficient in selective delivery to cancer cells overexpressing folate receptors. The improvement in yield of the targeting component can significantly facilitate “scale up” of folate receptor-mediated liposomal cancer therapy to the preclinical and clinical levels of investigations.