Synthesis and physicochemical characterization of folate-cyclodextrin bioconjugate for active drug delivery

beta-Cyclodextrin-poly(ethylene glycol)-folic acid conjugate (CD-PEG-FA) was synthesized according to a two-step procedure: (1). synthesis of CD-PEG-NH(2) by reaction of monotosyl-activated beta-cyclodextrin with excess of 700 Da diamino-PEG; (2). synthesis of CD-PEG-FA by reaction of CD-PEG-NH(2) with succinimidyl ester-activated folic acid. The CD-PEG-NH(2) intermediate was purified by precipitation in acetone, and the CD-PEG-FA by gel permeation and C-18 reversed-phase chromatography. Both CD-PEG-NH(2) and CD-PEG-FA were analyzed by mass spectrometry, (1)H NMR, and UV-vis spectroscopy. All analytical methods confirmed the theoretical composition of the conjugates: the CD-PEG-NH(2) intermediate was composed of CD and PEG in the molar ratio of 1:1, and the CD-PEG-FA was composed of beta-cyclodextrin, PEG, and folic acid in the molar ratio of 1:1:1. The CD-PEG-FA conjugate was highly soluble in buffer (>42 mM) as compared to the unmodified beta-cyclodextrin (16.3 mM). Phase solubility diagrams of beta-estradiol revealed that drug solubility increases from 11 microM in buffer to 600 microM in the presence of beta-cyclodextrins and 5900 microM with CD-PEG-FA. However, the affinity of beta-estradiol for beta-cyclodextrins decreased about 4 times with PEG and folic acid conjugation. Stability studies carried out using chlorambucil confirmed that the conjugate partially prevents drug degradation in buffer, although this effect was considerably lower than that obtained with beta-cyclodextrin. Computer modeling studies showed that the folic acid linked to the beta-cyclodextrins through a PEG spacer could partially interact with the cyclodextrin cavity. Finally, CD-PEG-FA displayed reduced hemolytic effect as compared to unmodified beta-cyclodextrin.     

The influence of extracellular folate concentration on methotrexate uptake by human KB cells. Partial characterization of a membrane-associated methotrexate binding protein

Methotrexate accumulation, subcellular distribution, metabolism, and cytotoxicity were studied in human epidermoid carcinoma (KB) cells that were exposed to a low extracellular concentration of methotrexate (25 nM) following culture in widely differing concentrations of folic acid. KB cells cultured in standard medium with a high folic acid concentration (2.3 microM) had high levels of cellular folate (21.4 pmol/10(6) cells). Five passages through low folate (2.7 nM) medium reduced the level of cellular folate to near physiologic levels (0.4-1.0 pmol/10(6) cells). In contrast to KB cells cultured in standard medium, in KB cells cultured in low folate medium, 1) methotrexate inhibited growth; 2) methotrexate uptake was markedly increased; 3) methotrexate polyglutamation was almost complete; 4) methotrexate binding to dihydrofolate reductase was markedly enhanced; and 5) significant methotrexate binding to a previously undescribed membrane-associated protein occurred. The amount of methotrexate bound to the membrane-associated protein from KB cells cultured in low folate medium equaled the quantities bound by dihydrofolate reductase. Further characterization of this membrane-associated protein indicated that it was soluble in solutions containing Triton X-100, was capable of binding folic acid as well as methotrexate, had an apparent Mr of 160,000 by gel filtration in the presence of Triton X-100, and was precipitated by antiserum to human placental folate receptor. This membrane-associated protein may play an important role in the uptake and metabolism of methotrexate under physiologic conditions.     

Use of a folate-PPE conjugate to image cancer cells in vitro

We have demonstrated synthesis and application of a water-soluble, folate-substituted poly(p-phenyleneethynylene) (PPE) as a fluorescent contrast agent to image cancer cells. This fluorescent polymer targets and images KB cancer cells in vitro with high selectivity. To deliver PPE to the cells, folate ligands have been attached to an amine-functionalized PPE via an amide coupling agent. The hydrolysis of the ester groups gave a water-soluble PPE, 5. The PPE 5 is minimally cytotoxic at concentrations of 1-10 microg/mL, which is sufficient to stain KB cancer cells efficiently. PPE 6, devoid of folate ligands, did not stain KB cells. As a low folic acid (-) receptor control group, NIH 3T3 fibroblast cells were incubated with 5 and did not show fluorescent labeling. The folate receptor-mediated endocytosis of KB cells was evidenced by laser scanning confocal microscopy and fluorescence microscopy. The photochemical stability and ability to sustain multivalency provide advantages of PPEs over other fluorescent contrast agents. Their minimal cytotoxicity makes the PPE superior to the cytotoxic CdSe quantum dots.     

Folate-conjugated iron oxide nanoparticles for solid tumor targeting as potential specific magnetic hyperthermia mediators: synthesis, physicochemical characterization, and in vitro experiments

New folate-conjugated superparamagnetic maghemite nanoparticles have been synthesized for the intracellular hyperthermia treatment of solid tumors. These ultradispersed nanosystems have been characterized for their physicochemical properties and tumor cell targeting ability, facilitated by surface modification with folic acid. Preliminary experiments of nanoparticles heating under the influence of an alternating magnetic field at 108 kHz have been also performed. The nanoparticle size, surface charge, and colloidal stability have been assessed in various conditions of ionic strength and pH. The ability of these folate "decorated" maghemite nanoparticles to recognize the folate receptor has been investigated both by surface plasmon resonance and in folate receptor expressing cell lines, using radiolabeled folic acid in competitive binding experiments. The specificity of nanoparticle cellular uptake has been further investigated by transmission electron microscopy after incubation of these nanoparticles in the presence of three cell lines with differing folate receptor expression levels. Qualitative and quantitative determinations of both folate nanoparticles and nontargeted control nanoparticles demonstrated a specific cell internalization of the folate superparamagnetic nanoparticles.     

Polyvalent saccharide-functionalized generation 3 poly(amidoamine) dendrimer-methotrexate conjugate as a potential anticancer agent

A saccharide-terminated generation 3 (G3) polyamidoamine (PAMAM) dendrimer was synthesized as a drug carrier. Utilizing this dendritic platform, we have successfully synthesized polyvalent conjugates (G3-MTX) containing the drug methotrexate (MTX). Surface Plasmon Resonance (SPR) results showed that G3-MTX presented three orders of magnitude enhancement in binding avidity to folate-binding protein (FBP) as compared to the free folic acid (FA). Flow cytometric and confocal microscopic analysis showed that conjugate (G3-MTX-FI) containing imaging agent fluorescein-5(6)-carboxamidohexanoic acid (FI) was internalized into folate receptor (FR)-expressing KB cells in dose-dependent and receptor-mediated fashion. The G3-MTX induced a dose-dependent cytotoxicity in the KB cells. Therefore, the polyvalent G3-MTX may have potential as an anticancer nanodevice for the specific targeting and killing of FR-expressing tumor cells.     

Folate-liposome-mediated antisense oligodeoxynucleotide targeting to cancer cells: evaluation in vitro and in vivo

The objective of this study was to investigate the use of folate-targeted liposomes for the delivery of encapsulated oligonucleotides to folate receptor (FR)-positive tumor cells in vitro and in vivo. This project involved the synthesis and biological evaluation of many folate-PEG-lipid conjugates, where the chemical form of the folate moiety (pteroate) and the length of the PEG linker chain were varied widely. Folate-targeted oligonucleotide-containing liposomes were prepared using conventional methods, and the extent of cell uptake was evaluated using, among others, the FR positive KB cell line. Oligonucleotide-loaded folate-targeted liposomes were found to rapidly associate with the KB cells, and saturation was typically reached within the first hour of incubation at 37 degrees C. Nearly 100,000 liposomes per cell were bound or internalized at saturation. Importantly, cell association was blocked by a large excess folic acid, thus reflecting the FR-specific nature of the cell interaction. Full targeting potential was achieved with PEG linkers as low as 1000 in molecular weight, and pteroates bearing glycine or gamma-aminobutyryl residues juxtaposed to the pteroic acid moiety were also effective for targeting, provided that a terminal cysteine moiety was present at the distal end of the PEG chain for added hydrophilicity. When tested in vivo, folate-targeted liposomes were found to deliver approximately 1.8-fold more oligonucleotide to the livers of nude mice (relative to the nontargeted PEG-containing formulations); however, no improvement in KB tumor uptake was observed. We conclude from these results that folate liposomes can effectively deliver oligonucleotides into folate receptor-bearing cells in vitro, but additional barriers exist in vivo that prevent or decrease effective tumor uptake and retention.     

Role of the membrane-associated folate binding protein (folate receptor) in methotrexate transport by human KB cells

The uptake of methotrexate by KB cells was observed to be dependent on time, temperature, and concentration of extracellular methotrexate. The Kd for methotrexate surface binding to KB cells was approximately 200 nM. Following exposure of KB cells to trace quantities of [3H]methotrexate for periods ranging from 6 min to 24 h, the cellular methotrexate was progressively formed into methotrexate polyglutamates and was bound to dihydrofolate reductase as well as to a particulate folate binding protein. To further study the mechanism of methotrexate uptake in KB cells, the N-hydroxysuccinimide ester of methotrexate was used to covalently label the surface of KB cells and to inhibit transport of methotrexate. The N-hydroxysuccinimide ester of methotrexate was bound to a species of protein with an apparent molecular weight of 160,000 in 1% (v/v) Triton X-100 that bound folic acid and was specifically precipitated by antiserum raised against the previously purified high-affinity folate binding protein (the folate receptor) from human KB cells. In addition, trypsin was utilized to remove surface-accessible covalently bound methotrexate. The amount of covalently bound methotrexate that could be released by trypsin initially decreased on incubation at 37 degrees C, suggesting that the methotrexate and binding protein were internalized. However, with time, trypsin could again release the covalently bound methotrexate, suggesting that the binding protein cycles from the external cell surface to the inside of the cell and out again.     

Synthesis and grafting of thioctic acid-PEG-folate conjugates onto Au nanoparticles for selective targeting of folate receptor-positive tumor cells

This paper reports the creation of Au nanoparticles (AuNP) that are soluble in aqueous solution over a broad range of pH and ionic strength values and that are capable of selective uptake by folate receptor positive (FR+) cancer cells. A novel poly(ethylene glycol) (PEG) construct with thioctic acid and folic acid coupled on opposite ends of the polymer chain was synthesized for targeting the AuNP to FR+ tumor cells via receptor-mediated endocytosis. These folic acid-PEG-thioctic acid conjugates were grafted onto 10-nm-diameter Au particles in aqueous solution. The resulting folate-PEG-coated nanoparticles do not aggregate over a pH range of from 2 to 12 and at electrolyte concentrations of up to 0.5 M NaCl with particle concentrations as high as 1.5 x 10(13) particles/mL. Transmission electron microscopy was used to document the performance of these coated nanoparticles in cell culture. Selective uptake of folate-PEG grafted AuNPs by KB cells, a FR+ cell line that overexpress the folate receptor, was observed. AuNP uptake was minimal in cells that (1) do not overexpress the folate receptor, (2) were exposed to AuNP lacking the folate-PEG conjugate, or (3) were co-incubated with free folic acid in large excess relative to the folate-PEG grafted AuNP. Understanding this process is an important step in the development of methods that use targeted metal nanoparticles for tumor imaging and ablation.     

Effects of folate receptor expression following stable transfection into wild type and methotrexate transport-deficient ZR-75-1 human breast cancer cells.

Two biochemically distinct systems, the high affinity folate receptor and the lower affinity reduced-folate carrier, have each been implicated in mediating the transport of folates and antifolates into cells. Previous studies from our laboratory have shown that methotrexate accumulation into wild type (WT) ZR-75-1 human breast cancer cells involves a system with characteristics of the reduced-folate carrier, that this system is deficient in methotrexate resistant (MTXR) ZR-75-1 cells in which methotrexate transport is undetectable and that neither breast cancer cell line expresses folate receptors. In this report we examined the possible interaction of the reduced-folate carrier with folate receptors by stably transfecting both WT ZR-75-1 and MTXR ZR-75-1 cells with an expression vector containing a folate receptor cDNA. Clones of stably transfected MTXR ZR-75-1 and WT ZR-75-1 cells expressing comparable levels of folate receptors were studied and compared to the nontransfected cell lines. Although nontransfected WT and MTXR ZR-75-1 cell lines require concentrations > or = 100 nM folic acid for growth, the expression of folate receptors in transfected WT and MTXR ZR-75-1 cells permitted the growth of both cell lines in low concentrations (1 nM) of folic acid. While the defect in the reduced-folate carrier system in MTXR ZR-75-1 cells inhibits their growth in medium containing low concentrations of folinic acid (< or = 1 microM), MTXR ZR-75-1 cells expressing folate receptors display uninhibited growth in 1 nM folinic acid. The accumulation of folic acid, folinic acid, and methotrexate is enhanced in folate receptor-transfected WT ZR-75-1 cells and MTXR ZR-75-1 cells. Furthermore, the accumulation of folates and antifolate was similar in both transfected WT and MTXR ZR-75-1 cell lines that expressed folate receptors. This suggests that alterations in the reduced-folate carrier do not affect folate receptor function. We also examined the effect of folate receptor expression on the sensitivity of WT and MTXR ZR-75-1 cells to methotrexate and to the lipophillic antifolate trimetrexate. Increased folate receptor expression decreased the sensitivity of WT ZR-75-1 cells toward the antifolate trimetrexate, presumably through increased uptake of reduced folates. Although the expression of the folate receptor enhanced the growth of both cell lines in low folate concentrations, it did not affect the sensitivity of either WT or MTXR ZR-75-1 cells to methotrexate.     

Receptor-Specific Delivery of Liposomes Via Folate-Peg-Chol

Abstract

A novel lipophilic conjugate of folate, folate-PEG-Chol, was synthesized and evaluated for receptor-mediated targeting of liposomes to tumor cells. Liposomes composed of DSPC/Chol/PEG-DSPE/folate-PEG-Chol (60/ 34/5/1, m/m) were taken up by cultured folate receptor-bearing KB cells via a saturable mechanism. Cellular binding of these liposomes could be competitively inhibited by free folic acid with an IC₅₀ of 0.39 mM, indicating an extraordinarily high binding affinity. Fluorescence micrographs of KB cells treated with targeted liposomes encapsulating calcein showed that they were distributed both on the cell surface and in intracellular vesicular compartments. Targeted liposomes carrying doxorubicin were shown to be 38 times more toxic to KB cells than non-targeted control liposomes. A biodistribution study in receptor-positive tumor-bearing C57BL/6 mice showed no significant differences between the tumor uptake of folate-PEG-liposomes and non-targeted control liposomes. This study has demonstrated that cholesterol could be used as an alternative to phospholipids as an effective anchor for incorporation of a targeting ligand into liposomes.     

Targeting folate receptor with folate linked to extremities of poly(ethylene glycol)-grafted liposomes: in vitro studies

Conjugates of three components, folic acid-poly(ethylene glycol)-distearoylphosphatidylethanolamine (FA-PEG-DSPE), derived from PEG with molecular masses of 2000 and 3350 Da were synthesized by a carbodiimide-mediated coupling of FA to H2N-PEG-DSPE. The conjugates were characterized by 1H NMR, MALDI-TOF, and HPLC analysis of enzymatic cleavage with carboxypeptidase G. As a prototype of a folate receptor (FR)-targeted system, the conjugates were formulated at 0.5 mol % phospholipid in hydrogenated phosphatidylcholine/cholesterol liposomes with or without additional methoxyPEG2000-DSPE. In vitro binding studies were performed with sublines of M109 (murine lung carcinoma) and KB (human epidermal carcinoma) cells each containing high and low densities of FR. FA-PEG-DSPE significantly enhanced liposome binding to tumor cells. The best binding was observed when FA-PEG liposomes contained no additional mPEG-lipid. In fact, our experiments showed that the presence of mPEG on liposomal surfaces significantly inhibited FA-PEG-liposome binding to FR. Increasing the molecular mass of the PEG tether from 2000 to 3350 Da improved the FR binding, particularly in the case of mPEG-coated liposomes. The FA-PEG liposomes bound to M109-HiFR cells very avidly as demonstrated by the inability of free FA (used in a 700-fold excess either at the beginning or at the end of the incubation) to prevent the cell binding. This is in contrast to the 5-10-fold lower cell binding activity of mPEG-FA compared to that of free FA, and likely to be related to the multivalent nature of the liposome-bound FA. Only 22% of FA-PEG3350 and 32% of FA-PEG3350/mPEG cell-associated liposomes could be removed by exposure to pH 3, conditions that dissociate FA-FR, suggesting that more than two-thirds of the bound liposomes were internalized during incubation for 24 h at 37 degrees C. FA-targeted liposomes also show enhanced nonspecific binding to extracellular tissue culture components, a phenomenon especially relevant in short incubation time experiments.     

Synthesis and biological evaluation of folate receptor-targeted boronated PAMAM dendrimers as potential agents for neutron capture therapy

Successful treatment of cancer by boron neutron capture therapy (BNCT) requires the selective delivery of (10)B to constituent cells within a tumor. The expression of the folate receptor is amplified in a variety of human tumors and potentially might serve as a molecular target for BNCT. In the present study we have investigated the possibility of targeting the folate receptor on cancer cells using folic acid conjugates of boronated poly(ethylene glycol) (PEG) containing 3rd generation polyamidoamine dendrimers to obtain (10)B concentrations necessary for BNCT by reducing the uptake of these conjugates by the reticuloendothelial system. First we covalently attached 12-15 decaborate clusters to 3rd generation polyamidoamine dendrimers. Varying quantities of PEG units with varying chain lengths were then linked to these boronated dendrimers to reduce hepatic uptake. Among all prepared combinations, boronated dendrimers with 1-1.5 PEG(2000) units exhibited the lowest hepatic uptake in C57BL/6 mice (7.2-7.7% injected dose (ID)/g liver). Thus, two folate receptor-targeted boronated 3rd generation polyamidoamine dendrimers were prepared, one containing approximately 15 decaborate clusters and approximately 1 PEG(2000) unit with folic acid attached to the distal end, the other containing approximately 13 decaborate clusters, approximately 1 PEG(2000) unit, and approximately 1 PEG(800) unit with folic acid attached to the distal end. In vitro studies using folate receptor (+) KB cells demonstrated receptor-dependent uptake of the latter conjugate. Biodistribution studies with this conjugate in C57BL/6 mice bearing folate receptor (+) murine 24JK-FBP sarcomas resulted in selective tumor uptake (6.0% ID/g tumor), but also high hepatic (38.8% ID/g) and renal (62.8% ID/g) uptake, indicating that attachment of a second PEG unit and/or folic acid may adversely affect the pharmacodynamics of this conjugate.     

Synthesis and activity of a folate peptide camptothecin prodrug

A folate receptor targeted camptothecin prodrug was synthesized using a hydrophilic peptide spacer linked to folate via a releasable disulfide carbonate linker. The conjugate was found to possess high affinity for folate receptor-expressing cells and inhibited cell proliferation in human KB cells with an IC(50) of 10nM. Activity of the prodrug was completely blocked by excess folic acid, demonstrating receptor-mediated uptake.     

Uptake and trafficking of fluorescent conjugates of folic acid in intact kidney determined using intravital two-photon microscopy

Intravital two-photon microscopy was used to follow the uptake and trafficking of fluorescent conjugates of folic acid in the rat kidney. Intravenously administered folate-linked dye molecules quickly filled the plasma volume but not cellular components of the blood. Glomerular filtration occurred immediately and binding to proximal tubule cells was seen within seconds. Fluorescence from a pH-insensitive conjugate of folic acid, folate Texas red (FTR), was readily observed on the apical surface of the proximal tubules and in multiple cellular compartments, but little binding or uptake could be detected in any other kidney cells. Fluorescence from a pH-sensitive conjugate of folic acid, folate fluorescein, was seen only on the apical surface of proximal tubule cells, suggesting that internalized folate conjugates are localized to acidic compartments. The majority of the FTR conjugate internalized by proximal tubules accumulated within a lysosomal pool, as determined by colocalization studies. However, portions of FTR were also shown by electron microscopy to undergo transcytosis from apical to basal domains. Additional studies with colchicine, which is known to depolymerize microtubules and interrupt transcytosis, produced a marked reduction in endocytosis of FTR, with accumulation limited to the subapical region of the cell. No evidence of cytosolic release of either folate conjugate was observed, which may represent a key difference from the cytosolic deposition seen in neoplastic cells. Together, these data support the argument that folate conjugates (and, by extrapolation, physiological folate) bind to the apical surface of proximal tubule cells and are transported into and across the cells in endocytic compartments. proximal tubule cell     

Targeted gadolinium-loaded dendrimer nanoparticles for tumor-specific magnetic resonance contrast enhancement

A target-specific MRI contrast agent for tumor cells expressing high affinity folate receptor was synthesized using generation five (G5) ofpolyamidoamine (PAMAM) dendrimer. Surface modified dendrimer was functionalized for targeting with folic acid (FA) and the remaining terminal primary amines of the dendrimer were conjugated with the bifunctional NCS-DOTA chelator that forms stable complexes with gadolinium (Gd III). Dendrimer-DOTA conjugates were then complexed with GdCl3 followed by ICP-OES as well as MRI measurement of their longitudinal relaxivity (T1 s(-1) mM(-1)) of water. In xenograft tumors established in immunodeficient (SCID) mice with KB human epithelial cancer cells expressing folate receptor (FAR), the 3D MRI results showed specific and statistically significant signal enhancement in tumors generated with targeted Gd(III)-DOTA-G5-FA compared with signal generated by non-targeted Gd(III)-DOTA-G5 contrast nanoparticle. The targeted dendrimer contrast nanoparticles infiltrated tumor and were retained in tumor cells up to 48 hours post-injection of targeted contrast nanoparticle. The presence of folic acid on the dendrimer resulted in specific delivery of the nanoparticle to tissues and xenograft tumor cells expressing folate receptor in vivo. We present the specificity of the dendrimer nanoparticles for targeted cancer imaging with the prolonged clearance time compared with the current clinically approved gadodiamide (Omniscan) contrast agent. Potential application of this approach may include determination of the folate receptor status of tumors and monitoring of drug therapy.     

Synthesis and biological evaluation of diethylenetriamine pentaacetic acid-polyethylene glycol-folate: a new folate-derived, (99m)Tc-based radiopharmaceutical

(99m)Technetium-labeled diethylenetriamine pentaacetic acid-polyethylene glycol-folate (DTPA-PEG-folate) was synthesized and tested as a radiopharmaceutical agent, which targeted the lymphatic system with metastatic tumor. Folic acid was reacted with H2N-PEG-NH2 to yield H2N-PEG-folate. After purification by anion-exchange chromatography, the product was reacted with cyclic DTPA. By removal of unreacted DTPA by size-exclusion chromatography, DTPA-PEG-Folate was obtained. Fluorescein-5-isothiocyanate (FITC)-labeled DTPA-PEG-folate and DTPA-PEG-OCH3 were prepared via a dicyclohexylcarbodiimide-mediated coupling. In vitro competitive binding test showed that the uptake of [125I] folic acid was inhibited by DTPA-PEG-folate and the 50% inhibitory concentration was 4.37 pmol/L (R2 = 0.9922). The relative affinity of DTPA-PEG-FITC was 0.18 for human folate receptor comparing with folic acid. In cultured tumor cells, uptake of fluorescence-labeled DTPA-PEG-folate was found to increase significantly in folate-deficient medium compared with that of untargeted DTPA-PEG-OCH3 and FITC-ethylenediamine. The competition with free folic acid blocked the cell uptake of DTPA-PEG-folate. These results confirmed the DTPA-PEG-folate entered into KB cells through the folate receptor endocytosis pathway in vitro. The radiolabeled yield of [(99m)Tc] DTPA-PEG-folate was in excess of 98%, and specific activities of 7.4 kBq (0.2 microCi/microg) were achieved. After subcutaneous injection, [(99m)Tc] DTPA-PEG-folate exhibited an initial increase and successive decline of accumulation in popliteal nodes in normal Wistar rats. Expect for the kidney, uptake by other tissues was rather low. In a normal rabbit imagine study, the lymphatic vessels were readily visualized by single-photon-emission computed tomography following subcutaneous injection of [(99m)Tc] DTPA-PEG-folate. In conclusion, the [(99m)Tc] DTPA-PEG-folate conjugate may have a potential as a lymphatic tumor-targeted radiopharmaceutical.     

Design, synthesis, and biological evaluation of folic acid targeted tetraphenylporphyrin as novel photosensitizers for selective photodynamic therapy

Photodynamic therapy (PDT) is a cancer treatment involving systemic administration of a tumor-localizing photosensitizer; this, when activated by the appropriate light wavelength, interacts with molecular oxygen to form a toxic, short-lived species known as singlet oxygen, which is thought to mediate cellular death. Targeted PDT offers the opportunity of enhancing photodynamic efficiency by directly targeting diseased cells and tissues. Two new conjugates of three components, folic acid/hexane-1,6-diamine/4-carboxyphenylporphyrine 1 and folic acid/2,2'-(ethylenedioxy)-bis-ethylamine/4-carboxyphenylporphyrine 2 were synthesized. The conjugates were characterized by 1H NMR, MALDI, UV-visible spectroscopy, and fluorescence quantum yield. The targeted delivery of these photoactive compounds to KB nasopharyngeal cell line, which is one of the numerous tumor cell types that overexpress folate receptors was studied. It was found that after 24 h incubation, conjugates 1 and 2 cellular uptake was on average 7-fold higher than tetraphenylporphyrin (TPP) used as reference and that 1 and 2 cellular uptake kinetics increased steadily over the 24 h period, suggesting an active transport via receptor-mediated endocytosis. In corresponding results, conjugates 1 and 2 accumulation displayed a reduction of 70% in the presence of a competitive concentration of folic acid. Survival measurements demonstrated that KB cells were significantly more sensitive to conjugated porphyrins-mediated PDT. Under the same experimental conditions and the same photosensitizer concentration, TPP displayed no photocytotoxicity while conjugates 1 and 2 showed photodynamic activity with light dose values yielding 50% growth inhibition of 22.6 and 6.7 J/cm2, respectively.     

Clustering of mutations in the first transmembrane domain of the human reduced folate carrier in GW1843U89-resistant leukemia cells with impaired antifolate transport and augmented folate uptake

We have studied the molecular basis for the resistance of human CEM leukemia cells to GW1843, a thymidylate synthase inhibitor. GW1843-resistant cells displayed a approximately 100-fold resistance to GW1843 and methotrexate but were collaterally sensitive to the lipophilic antifolates trimetrexate and AG337, which enter cells by diffusion. These cells exhibited a 12-fold decreased methotrexate influx but surprisingly had a 2-fold decreased folic acid growth requirement. This was associated with a 4-fold increased influx of folic acid, a 3.5-fold increased steady-state level of folic acid, and a 2.3-fold expansion of the cellular folate pool. Characterization of the transport kinetic properties revealed that GW1843-resistant cells had the following alterations: (a) 11-fold decreased transport K(m) for folic acid; (b) 6-fold increased transport K(m) for GW1843; and (c) a slightly increased transport V(max) for folic acid. Sequence analysis showed that GW1843-resistant cells contained the mutations Val-29 --> Leu, Glu-45 --> Lys, and Ser-46 --> Ile in the first transmembrane domain of the reduced folate carrier. Transfection of the mutant-reduced folate carrier cDNA into methotrexate transport null cells conferred resistance to GW1843. This is the first demonstration of multiple mutations in a confined region of the human reduced folate carrier in an antifolate-resistant mutant. We conclude that certain amino acid residues in the first transmembrane domain play a key role in (anti)folate binding and in the conferring of drug resistance.     

Simultaneous activity of two different mechanisms of folate transport in ovarian carcinoma cell lines

We investigated whether the folate receptor α-isoform (FRα), which is overexpressed on ovarian carcinoma cells, is functionally active in internalizing the physiological form of folate, 5-methyl tetrahydrofolate (THF). Six ovarian tumor cell lines, expressing different levels of FRα (COR ≫ OVCAR3 > IGROV1 > OVCAR4 > SKOV3 > OVCAR5), were maintained in folate-depleted medium and internalization of 10 nM evaluated as acid-resistant radioactivity at 0° and 37°C. The amount of 5-methyl[³H]THF present in this fraction was not strictly related to the number of membrane receptors, since even cell lines with low FRα expression, e.g., OVCAR4, showed efficient internalization. Time-course studies indicated that, whereas no uptake was detected at 0°C, at 37°C the internalized fraction showed a slow and constant increase, until 4 h. At this time, the internalized radioactivity represented <50% of the total bound in COR, OVCAR3 and IGROV1 cells, whereas the other cell lines tested internalized fourfold more folate than their surface binding capacity. The incubation in the presence of a concentration (50 nM) of 5-methyl[³H]THF, which best ensures receptors saturation on cells with highest FR levels (COR and OVCAR3), had slight effect on surface binding of all the tested cell lines, including IGROV1 and SKOV3. In contrast, the increase of the uptake was more pronounced, particularly in SKOV3 cells. These results, together with the accumulation curves of folic acid (FA) and 5-methylTHF at 37°C, suggested the presence of a molecule on ovarian carcinoma cells with high affinity for reduced folates, possibly a reduced folate carrier (RFC). Measurement of radioactivity present in the supernatant of IGROV1 and SKOV3 cells, subjected to hypotonic lysis and cell fractionation, further indicated that 5-methyl[³H]THF was translocated to the cytosol and, despite differences in membrane levels of FRα expression this internalized fraction was similar in both cell lines. Inhibition experiments to selectively block FRα or RFC activity showed a differential sensitivity of the two pathways depending on the cell line examined. Internalization was more consistently inhibited on IGROV1 than on SKOV3 cells by treatments that disrupt FRα activity, e.g., incubation with excess FA and phosphatidylinositol specific phospholipase C, whereas Probenecid, which preferentially inhibits the carrier-mediated pathway, showed a strong inhibitory effect on both cell lines. These findings suggest that the internalization of 5-methylTHF in these tumor cells depends not only on the level of overexpressed FRα, but another transport route, with features characteristic for RFC, is functional and participates in folate uptake. J. Cell. Biochem. 65:479–491. © 1997 Wiley-Liss Inc.     

Suppressing unspecific cell uptake for targeted delivery using hydroxyethyl starch nanocapsules

Synthesizing nanocarriers with stealth properties and delivering a "payload" to the particular organ remains a big challenge but is the prime prerequisite for any in vivo application. As a nontoxic alternative to the modification by poly(ethylene glycol) PEG, we describe the synthesis of cross-linked hydroxyethyl starch (HES, M(w) 200,000 g/mol) nanocapsules with a size range of 170-300 nm, which do not show nonspecific uptake into cells. The specific uptake was shown by coupling a folic acid conjugate as a model targeting agent onto the surface of the nanocapsules, because folic acid has a high affinity to a variety of human carcinoma cell lines which overexpress the folate receptor on the cell surface. The covalent binding of the folic acid conjugate onto HES capsules was confirmed by FTIR and NMR spectroscopy. The coupling efficiency was determined using fluorescence spectroscopy. The specific cellular uptake of the HES nanocapsules after folic acid coupling into the folate-receptor presenting cells was studied by confocal laser scanning microscopy (CLSM) and flow cytometry.