Haloperidol-associated Stealth Liposomes
2005; Elsevier BV; Volume: 280; Issue: 16 Linguagem: Inglês
10.1074/jbc.m409723200
ISSN1083-351X
AutoresAmarnath Mukherjee, Tekkatte Krishnamurthy Prasad, Nalam Madhusudhana Rao, Rintu Banerjee,
Tópico(s)Nicotinic Acetylcholine Receptors Study
ResumoSigma receptors are membrane-bound proteins that are overexpressed in certain human malignancies including breast cancer. These receptors show very high affinity for various sigma ligands including neuroleptics like haloperidol. We hypothesized that in associating haloperidol-linked lipid into the cationic lipid-DNA complex, we can specifically target and deliver genes to breast cancer cells that overexpress sigma receptors. In the present study, haloperidol was chemically modified to conjugate at the distal end of the polyethylene glycollinked phospholipid, which was then incorporated into the cationic liposome known to condense and deliver genes inside cells. The resulting haloperidol-conjugated targeted lipoplex showed at least 10-fold higher (p < 0.001) reporter gene expression in MCF-7 cells than control lipoplex. The reporter gene expression of the targeted lipoplex was significantly blocked by haloperidol (p < 0.001) and by another sigma ligand, 1,3-ditolylguanidine (p < 0.001) in the majority of cationic lipid to DNA charge ratios (±). Spironolactone-mediated sigma receptor down-regulation enabled MCF-7 to show 10-fold lower transgene expression with targeted lipoplex compared with that obtained in spironolactone-untreated cells. The targeted lipoplex generated nonspecific gene expression in sigma receptor-nonexpressing human cancer cells such as Hela, KB, HepG2, and Chinese hamster ovary cells. Moreover, the transgene expression remained unabated in physiologically relevant serum concentrations. This is the first study to demonstrate that haloperidol-targeted gene delivery systems can mediate efficient targeting of genes to sigma receptor-overexpressing breast cancer cells, thereby becoming a novel class of therapeutics for the treatment of human cancers. Sigma receptors are membrane-bound proteins that are overexpressed in certain human malignancies including breast cancer. These receptors show very high affinity for various sigma ligands including neuroleptics like haloperidol. We hypothesized that in associating haloperidol-linked lipid into the cationic lipid-DNA complex, we can specifically target and deliver genes to breast cancer cells that overexpress sigma receptors. In the present study, haloperidol was chemically modified to conjugate at the distal end of the polyethylene glycollinked phospholipid, which was then incorporated into the cationic liposome known to condense and deliver genes inside cells. The resulting haloperidol-conjugated targeted lipoplex showed at least 10-fold higher (p < 0.001) reporter gene expression in MCF-7 cells than control lipoplex. The reporter gene expression of the targeted lipoplex was significantly blocked by haloperidol (p < 0.001) and by another sigma ligand, 1,3-ditolylguanidine (p < 0.001) in the majority of cationic lipid to DNA charge ratios (±). Spironolactone-mediated sigma receptor down-regulation enabled MCF-7 to show 10-fold lower transgene expression with targeted lipoplex compared with that obtained in spironolactone-untreated cells. The targeted lipoplex generated nonspecific gene expression in sigma receptor-nonexpressing human cancer cells such as Hela, KB, HepG2, and Chinese hamster ovary cells. Moreover, the transgene expression remained unabated in physiologically relevant serum concentrations. This is the first study to demonstrate that haloperidol-targeted gene delivery systems can mediate efficient targeting of genes to sigma receptor-overexpressing breast cancer cells, thereby becoming a novel class of therapeutics for the treatment of human cancers. Haloperidol is a common neuroleptic drug that is subtype non-selective yet shows a strong affinity for sigma receptors. Haloperidol and other sigma ligands have been shown to elicit various physiological processes, which include triggering apoptosis in cells of neuronal origin and in rapidly proliferating cells (1Vilner B.J. Bowen W.D. Eur. J. Pharmacol. 1993; 244: 199-201Crossref PubMed Scopus (80) Google Scholar, 2Vilner B.J. de Costa B.R. Bowen W.D. J. Neurosci. 1995; 15: 117-134Crossref PubMed Google Scholar, 3Brent P.J. Pang G. Little G. Dosen P.J. Van Helden D.F. Biochem. Biophys. Res. Commun. 1996; 219: 219-226Crossref PubMed Scopus (60) Google Scholar, 4Crawford K.W. Bowen W.D. Cancer Res. 2002; 62: 313-322PubMed Google Scholar). It is evident now that these physiological processes are exerted through haloperidol-sigma receptor interaction. Sigma receptors are membrane-bound protein receptors that are expressed in normal tissues, such as liver, endocrine glands, kidneys, lungs, gonads, central nervous system, and ovaries at basal levels (5Wolfe S Jr A. Culp S.G. De Souza E.B. Endocrinology. 1989; 124: 1160-1172Crossref PubMed Scopus (165) Google Scholar, 6Hellewell S.B. Bruce A. Feinstein G. Orringer J. Williams W. Bowen W.D. Eur. J. Pharmacol. 1994; 268: 9-18Crossref PubMed Scopus (414) Google Scholar). Although the physiological roles of these receptors in normal tissues are not yet clear, a diverse set of human tumors, such as melanoma, non-small cell lung carcinoma, breast tumors of neural origin, and prostate cancer overexpress sigma receptors (7Vilner B.J. John C.S. Bowen W.D. Cancer Res. 1995; 55: 408-413PubMed Google Scholar, 8John C.S. Bowen W.D. Saga T. Kinuya S. Vilner B.J. Baumgold J. Paik C.H. Reba R.C. Neumann R.D. Varma. V.M. J. Nucl. Med. 1993; 34: 2169-2175PubMed Google Scholar, 9John C.S. Vilner B.J. 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Cancer Res. 1999; 59: 4578-4583PubMed Google Scholar, 13John C.S. Bowen W.D. Fisher S.J. Lim B.B. Geyer B.C. Vilner B.J. Wahl R.L. Nucl. Med. Biol. 1999; 26: 377-382Crossref PubMed Scopus (39) Google Scholar). Liposomes encapsulating biologically active molecules have long been used as a vehicle to target the payload in vivo. In cancer-targeting studies, specific targeting to tumor cells by liposomes has previously been demonstrated (14Huang A. Huang L. Kennel S.J. J. Biol. Chem. 1980; 255: 8015-8018Abstract Full Text PDF PubMed Google Scholar, 15Huang A. Kennel S.J. Huang L. J. Biol. Chem. 1983; 258: 14034-14040Abstract Full Text PDF PubMed Google Scholar). Also immunoliposomes associated with PEG 1The abbreviations used are: PEG, polyethylene glycol; DODEAC, N,N-di-n-tetradecyl-N,N-(2-hydroxyethyl)ammonium chloride; DSPE-PEG(2000)-mal, distearoyl-sn-glycero-phosphatidylethanolamine-[ω-maleimido-polyethylene glycol(2000)]; DSPE-PEG-HP, distearoylglycerolphosphatidylethanolamine-polyethylene glycol(2000)-ω-carboxamido[2-(O′-haloperidolyl)]ethylcarboxylate; DTG, 1,3-o-ditolyl guanidine; HP, haloperidol; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PBS, phosphate-buffered saline; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; CHO, Chinese hamster ovary; N-Boc, N-butyloxycarbonyl; CMV, cytomegalovirus. have previously shown stealth property and tumor-targeting efficiency in vivo (16Ho R.J.Y. Rouse B. Huang L. Biochemistry. 1986; 25: 5500-5506Crossref PubMed Scopus (111) Google Scholar, 17Maruyama, K., Kennel, S. J., and Huang, L. Proc. Natl. Acad. Sci. U. S. A. 87, 5744–5748Google Scholar, 18Mori A. Kennel S.J. Huang L. Pharm. Res. 1993; 10: 507-514Crossref PubMed Scopus (41) Google Scholar). Recently, a non-immunogenic, small molecular weight sigma ligand carrying an anisamide moiety was used as a targeting ligand in a drug-carrying liposomal system to target anticancer drugs to sigma receptor-expressing prostate cancer cells in the xenograft tumor mice model (19Banerjee R. Tyagi P. Li S. Huang L Int. J. Cancer. 2004; 112: 693-700Crossref PubMed Scopus (218) Google Scholar). The above-mentioned study propelled us to investigate the use of a readily available inexpensive generic drug, haloperidol, which shows very high affinity for sigma receptors, as a targeting ligand in a stealth liposomal system carrying genes to sigma receptor-overexpressing breast adenocarcinoma cells. Without potentially reducing targeting ability, the haloperidol is chemically conjugated to phospholipids with a PEG spacer in between. This ligand-conjugated PEG-lipid is included in 5 mol %, along with a known cationic lipid (20Singh R.S. Mukherjee K. Banerjee R. Chaudhuri A. Hait S.K. Moulik S. Ramdas Y. Vijayalakshmi A. Rao N.M. Chem. Eur. J. 2002; 8: 900-909Crossref PubMed Scopus (49) Google Scholar) and cholesterol, to form long-lived circulating and targeted cationic liposomes. These cationic liposomes pre-condense plasmid DNA containing the reporter or therapeutic gene. It is expected that haloperidol having high affinity for the sigma receptor, upon incorporation into the surface of the liposome, will avidly interact with cells expressing the sigma receptor. This liposome-cell interaction eventually ferries the genetic cargo inside the cell, possibly through receptor-mediated uptake. As an added advantage, the liposome-DNA complex will remain stabilized because PEG is known to prolong the circulation times of the liposome in vivo (21Klibanov A.L. Maruyama K. Torchilin V.P. Huang L. FEBS Lett. 1990; 268: 235-237Crossref PubMed Scopus (1774) Google Scholar, 22Zalipsky S. Brandeis E. Newman M.S. Woodle M.C. FEBS Lett. 1994; 353: 71-74Crossref PubMed Scopus (96) Google Scholar). Moreover, because of the low mol % of haloperidol-associated lipid, the lipoplex is expected to exert minimal agonistic effect, if any, to sigma receptor-expressing cells. To this end, we have incorporated haloperidol-conjugated PEG phospholipid in the cationic liposome formulation and condense reporter plasmid DNA. The targeted uptake followed by the efficiency of reporter gene expression in sigma receptor-overexpressing human breast carcinoma cell MCF-7 was studied in the presence of serum-containing medium and in the absence or presence of free haloperidol and ditolylguanidine. Chemicals and General Procedures—Phospholipids such as DSPE-PEG(2000)-maleimide and DSPE-PEG(2000)-COOH were purchased from Avanti Polar Lipids (Birmingham, AL). Cholesterol, trypsin, EDTA, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), dimethyl sulfoxide (Me2SO), and haloperidol (HP) were purchased from Sigma. All the chemicals and organic solvents required for synthesis were purchased from either Aldrich (Milwaukee, WI) or S.D. Fine Chem (Mumbai, India). They were used without further purification. Spironolactone was obtained from the drug aldactone (RPG Life Sciences Ltd., Ankleshwar, India). Briefly, aldactone tablets (50 mg by spironolactone weight) were crushed and dissolved in 10 ml of water. The drug was extracted by dichloromethane (2 × 25 ml). Upon evaporation of the non-aqueous layer, the free drug was crystallized out in methanol at –20 °C. The purity and authenticity of the crystallized compound (white needle, 48 mg) was characterized by TLC, melting point analysis, and by its NMR spectrum. 1H NMR spectra were recorded on a Bruker FT 300 MHz and Varian FT 200 MHz and 400 MHz instrument. Cell Culture—MCF-7, CHO, Hela, KB, and HepG2 cells were purchased from the National Center for Cell Sciences (Pune, India) and were mycoplasma-free. Cells were cultured in DMEM (ATCC) containing 10% fetal bovine serum (Sigma) and 1% penicillin-streptomycin at 37 °C in a humidified atmosphere of 5% CO2 in air. Cultures of 85–90% confluency were used for all of the experiments. The cells were trypsinized, counted, and subcultured in 96-well plates for transfection and viability studies. The cells were allowed to adhere overnight before they were used for experiments. Synthesis of Ligands—The synthetic procedure for preparing the lipid DSPE-PEG-HP is depicted schematically in Fig. 1. Detailed experimental procedures are delineated below. Step a: Synthesis of N-Boc-β-Alanine-Haloperidol Conjugate (Compound I,Fig. 1)—A mixture of N-Boc β-alanine (400 mg, 2.1 mmol), haloperidol (400 mg, 1 mmol), and N,N-dimethylaminopyridine (DMAP, 20 mg, catalytic) were mixed in a 25-ml round bottom flask in 5 ml of dry DCM and stirred in ice for 0.5 h. To the mixture, EDC (240 mg, 1.2 mmol) was added, and the sample was stirred in an ice bath for 1 h. The reaction mixture was further stirred for 12 h at room temperature. The reaction mixture was dissolved in 20 ml of dichloromethane, washed with water (2 × 20 ml), and brine (1 × 20 ml), and dried with anhydrous Na2SO4. Column chromatographic purification (using 60–120 mesh silica gel and 2% methanol-chloroform as eluent) of the residue yielded compound I, a white solid (116 mg, 20% yield, Rf 0.6 in 5% methanol/chloroform). 1H NMR (200 MHz, CDCl3): δ = 1.2 [s, 9H, -(CH3)3C-O-CONH], 1.9 [t, 4H, -CH2-CH2-N-CH2-CH2], 2–2.6 [m, 8H, (CH2)2-N-CH2-CH2-], 2.7 [m, 2H, -O-CO-CH2-CH2-NHBOC], 2.9 [t, 2H, -CH2-COAr], 3.2 [m, 2H, -CH2NHBOC], 4.8–4.9 [bs, 1H, BOC-NH], 6.9–7.2 [m, 6H, o- and m-C6H4-F + o-C6H4-Cl, 8 [m, 2H, o-C6H4-CO]. FABMS (LSIMS): m/z, 547 [M+] for C29H36O5N2ClF. Step b: Boc Deprotection of N-Boc-β-Alanine-Haloperidol Conjugate (Compound II,Fig. 1)—N-Boc-β-alanine-haloperidol conjugate, compound I (intermediate product obtained from step a, 280 mg, 0.6 mmol) was put into a 25-ml round bottom flask and dissolved in 5 ml of 10% trifluoroacetic acid-dichloromethane (v/v). The reaction mixture was stirred over an ice bath for 2 h, and then the solution was neutralized using saturated NaHCO3 solution. The mixture was extracted with DCM (2 × 15 ml), and the organic layer was dried with Na2SO4. Evaporation of the organic layer afforded compound II as a gummy material (160 mg, 70% yield, Rf 0.1 in 5% methanol/chloroform, active in ninhydrin charring). Because compound II was obtained as ∼95% pure (revealed by TLC), it was directly used for the final step. Step c: Synthesis of DSPE-PEG-HP—A mixture of DSPE-PEG(2000)-COOH (50 mg, 0.018 mmol) and compound II (30 mg, 0.06 mmol) were put into a 10-ml round bottom flask in 3 ml of dry DCM and stirred over an ice bath. After 0.5 h, dicyclohexylcarbodiimide (DCC, 5 mg, 0.023 mmol) was added to the reaction, with continued stirring at room temperature for 12 h. The solvent was evaporated, and the crude product was purified three times by recrystallization using methanol/ether (1:15 v/v) as solvent. The purified compound obtained as a white gummy material (38 mg, 66% yield with respect to the PEG-lipid, Rf 0.1 at CHCl3/acetone/methanol 79:20:1, UV active). 1H NMR (400 MHz, CD3OD) of representative peaks: δ = 0.9 [t, 6H, O-CO-CH2-(CH2)14CH2CH3, 1.2–1.6 [m, 56H, O-CO-CH2-(CH2)14CH2CH3]], 7.2 [m, 2H, & o-C6H4Cl], 7.4 [m, 4H, o- and m-C6H4F], 8.2 [m, 2H, o-C6H4-CO]. The integration of the protons in the aromatic moiety when attached to the high molecular weight PEG does not give accurate peak heights in contrast to other protons in NMR, as reported elsewhere (23Greenwald R.B. Gilbert C.W. Pendri A. Conover C.D. Xia J. Martinez A. J. Med. Chem. 1996; 39: 424-431Crossref PubMed Scopus (242) Google Scholar). Electrospray mass spectra of the product revealed an inverted U shape mass spectral pattern with an increment of 44 (a characteristic of PEG-based molecules) spanning between molecular weights 2922 and 3450. The peak of the mass spectral pattern is obtained at molecular weight 3149 (possibly M+2). The starting material DSPE-PEG-COOH shows a pattern spanning between molecular weights 2630 and 2985, with a peak at molecular weight 2718. The peak for the product was considered 3147 [2718 + 429 (contributed from HP)] and was accepted as the molecular weight of the product for the mole calculations hereafter. Liposome Preparations—The lipid films were prepared by drying the chloroform solution, from a total of 2.05 μmol of DODEAC, cholesterol and DSPE-PEG-HP or DSPE-PEG-mal under a gentle stream of N2, under vacuum for at least 6 h. The lipid mixtures were composed of DODEAC/Chol/DSPE-PEG-HP or DODEAC/Chol/DSPE-PEG-mal in a molar ratio of 1:1:0.05. The mixture was hydrated with 1 ml of sterile water overnight and then first subjected to a low intensity bath sonication for 15 min at room temperature and then probe sonication for 2 min in ice using a constant duty cycle and output control magnitude of 2–3 in a Branson Sonifier 450. DNA Binding Assay—The DNA binding ability of the targeted and non-targeted lipids containing DSPE-PEG-HP and DSPE-PEG-mal, respectively, was assessed by gel retardation assay on a 0.8%-agarose gel. 0.40 μg of pCMV-SPORT-β-gal was complexed with the cationic lipids (at a cationic lipid/DNA charge ratio 8:1, 4:1, 2:1, and 1:1) in a total volume of 16 μl of HEPES buffer (pH 7.4) and incubated at room temperature for 30 min on a rotary shaker. 3 μl of 6× loading buffer (0.25% bromphenol blue, 40% sucrose) was added, and the total solution was loaded to each well. The samples were electrophoresed at 80 V for ∼2 h, and the DNA bands were visualized by staining for 30 min with ethidium bromide solution followed by 30 min of destaining in water. DNase 1 Sensitivity Assay—In a typical assay, 3 nmol of DNA (1 μg) were complexed with both targeted and non-targeted cationic lipids containing DSPE-PEG-HP and DSPE-PEG-mal, respectively, in a (±) charge ratio of 8:1, 4:1, 2:1, and 1:1. The mixture was incubated at room temperature for 30 min on a rotary shaker. Subsequently, the complex was treated with DNase I (at a final concentration of 10 ng/3 nmol of pDNA), in the presence of 20 mm MgCl2. The volume was brought up to 50 μl with HEPES buffer (pH 7.4) and incubated at 37 °C for 0.5 h. To stop the hydrolysis reaction, EDTA was added to a final concentration of 20 mm, and the mixture was incubated at 60 °C for 10 min in a water bath. The aqueous layer was extracted with 50 μl of phenol/chloroform mixture (1:1, v/v) and subsequently centrifuged at 10,000 rpm for 5 min. The aqueous supernatant was separated, loaded (20 μl) on a 0.8% agarose gel, and electrophoresed at 80 V for 3 h. DNase I-treated and untreated naked DNA were also included in the same experiment. The bands were visualized after 45 min of ethidium bromide staining followed by 30 min of destaining in water. Western Blot Analysis of Sigma Receptors—MCF-7 and CHO cells were grown in 75-cm2 flasks until they reached a confluency of about 80%. Cells were detached from the flasks using 0.1% EDTA solution. Whole cell lysates were prepared by directly lysing the cells in SDS loading buffer. Total protein content in each sample was determined by amido black staining (24Sheffield J.B. Graff D. Li H.P. Anal. Biochem. 1987; 166: 49-54Crossref PubMed Scopus (100) Google Scholar). Equal amounts of protein were loaded and separated on a 15% SDS-PAGE. Proteins were transferred onto nitrocellulose membrane (Hybond-C extra, Amersham Biosciences) using semidry blotting. Gels were stained with Coomassie Brilliant Blue R-250 to visualize equal loading of protein samples. The membrane was blocked overnight at 4 °C with 5% BLOTTO (Santa Cruz Biotechnology). The blot was then incubated with polyclonal antibody raised against full-length recombinant sigma receptor 1 of human origin in rabbit (Santa Cruz Biotechnology) at 1:500 dilution for 1 h at room temperature. After washing, the membrane was incubated with goat anti-rabbit secondary antibody conjugated to horseradish peroxidase (Amersham Biosciences) at 1:4000 dilution for 45 min. To confirm equal loading of protein samples, the blot was also probed for levels of β-actin using monoclonal antibody (Sigma) raised in the mouse against β-actin at 1:800 dilution. We used anti-mouse horseradish peroxidase-conjugated secondary antibody (Amersham Biosciences) to detect actin. Using the enhanced chemiluminescence method and exposing the membrane to x-ray film (Eastman Kodak Company) in a dark room, protein bands were visualized. Gene Transfection—Cells were seeded at a density of 12,000 cells/well in a 96-well plate usually 18–24 h before transfection. 0.30 μg of pCMV-SPORT-β-gal DNA (diluted to 50 μl with DMEM) was complexed with varying amount of cationic liposomes (diluted to 50 μl with plain DMEM) for 30 min. The molar ratios (lipid/DNA) were 8:1, 4:1, 2:1, and 1:1. After complexation was completed, 200 μl of DMEM containing 10% FBS (CM1X) were added to the resulting lipoplexes for triplicate experiments. Thus the final concentration of serum became 6.7%. Cells were washed with phosphate-buffered saline (PBS), pH 7.4 (1 × 200 μl) and then with lipoplex (100 μl). After incubation of the cell plates in a humidified atmosphere containing 5% CO2 at 37 °C for 4 h, 100 μl of DMEM containing 10% FBS (CM1X) were added to cells. The reporter gene activity was assayed after 48 h. The medium was removed completely from the wells, and cells were lysed with 50 μl of 1× reporter lysis buffer (Promega) for 30 min. The β-galactosidase activity per well was estimated by adding 50 μlof2× substrate (1.33 mg/ml of ONPG, 0.2 m sodium phosphate, pH 7.3, and 2 mm magnesium chloride) to the cell lysate in the 96-well plate. Absorption of the product ortho-nitrophenol at 405 nm was converted to absolute β-galactosidase units using a calibration curve constructed with commercial β-galactosidase enzyme. For the haloperidol- and DTG-pretreated experiments, cells (in 100 μl of complete medium) were treated either with haloperidol or DTG (in Me2SO) in a final concentration of 20 and 100 μm, respectively, for 2 h. Cells were incubated in a humidified atmosphere containing 5% CO2 at 37 °C. Medium was removed, and the cells were washed with PBS (1 × 100 μl). The cells were subsequently treated with the lipoplexes, and the reporter gene assay was performed according to the above-mentioned procedure. For studies with spironolactone-treated MCF-7 cells, the cells were treated with spironolactone at a final concentration of 10 μm in serum medium for 1 day. Microscopically, cells show no change in cellular morphology compared with spironolactone-untreated MCF-7 cells before plating. The cells upon becoming 80–90% confluent were plated in 96-well plates in the presence of spironolactone. The cells were kept in spironolactone-containing medium overnight. The medium was removed, and cells were washed with PBS (1 × 100 μl) before lipoplex was added to the cells. The transfection and assay were performed as mentioned before. For the serum dependence study, upon incubation for 30 min in DMEM, 200 μl of either of the following were added to the lipoplex: (a) DMEM, (b) 10% FBS-containing DMEM, (c) 40% FBS-containing DMEM, (d) 80% FBS-containing DMEM, or (e) 100% FBS. 100 μl of each of the 300 μl of resulting complex solution were added to each of the wells of the triplicate experiments, and the cells were incubated for 4 h as described above. The final percentages of serum were 0, 26, 53, and 66, respectively, during incubation. After 4 h the medium was removed, and the cells were incubated in 10% FBS-containing DMEM and were incubated for another 48 h. The cellular assays were done as described previously. The transfection values are reported as the average values of triplicate experiment performed in the same plate on the same day. To verify reproducibility, each transfection experiment in the MCF-7 cell was performed at least four times on four different days. Other cell line data were representative data from at least two transfection experiments. The day to day variations in transfection efficiency were mostly within 2–3-fold and were dependent on the condition of cells. Cell Viability Assay—Cytotoxicity of the cationic lipids was assessed using the MTT reduction assay as described earlier (25Hansen M-B. Neilson S.E. Berg K. J. Immunol. Methods. 1989; 119: 203-210Crossref PubMed Scopus (3343) Google Scholar). The cytotoxicity assay was performed in 96-well plates, keeping the ratio of number of cells to amount of cationic lipid constant as was maintained in previously described transfection experiments. Briefly, 4 h after the addition of lipoplexes, MTT (5 mg/ml PBS) was added to the cells and was incubated for 3–4 h at 37 °C. Results are expressed as shown in Equation 1. Percent viability=[A550(treated cells)-background]/[A550(untreated cells)-background]×100 eq.1 Statistical Analysis—Data were expressed as mean ± S.D. and statistically analyzed by the two-tailed unpaired Student's t test using Microsoft Excel (Seattle, WA). Data were primarily considered significant if p < 0.001. Synthesis of DSPE-PEG-HP—HP is a very potent sigma drug, which possesses excellent affinity toward both subtypes of sigma receptors. With the help of the β-alanine adduct, HP is suitably modified to conjugate it to the PEG-grafted phospholipid, wherein PEG (Mr 2000) acts as spacer between the lipid and the targeting ligand. The inclusion of the PEG spacer is done to improve the targetability and to provide stealth property to the liposome containing the PEG-lipid. Fig. 1 shows the scheme for the synthesis of haloperidol-derivatized phospholipid. The overall yield for the reactions involved in the suitable modification of HP is 60%, and the final conjugation yield is 70%. Binding Study and DNase I Treatment Study of DNA—The targeted and non-targeted lipoplexes showed practically no difference in their binding property (Fig. 2A). Both the liposomes exhibit comparable affinity and bind to DNA in a similar fashion. At and beyond the 4:1 (±) charge ratio, DNA remains completely complexed with PEG-associated cationic liposomes, whereas at and below the 2:1 (±) charge ratio, a majority of the DNA remains non-complexed. These binding properties are corroborated in the DNase I treatment study of lipoplex (Fig. 2B). The lipoplexes irrespective of the presence of targeted or non-targeted PEG-lipid save DNA from DNase I with similar avidity in a ± charge ratio of 4:1 or greater. Targeted Gene Transfection in MCF-7 Cells—Fig. 3A shows the comparison between reporter gene expression in HP- and DTG-untreated sigma receptor-overexpressing breast adenocarcinoma MCF-7 cells treated with cationic lipoplex, which contain either targeted lipid DSPE-PEG-HP or non-targeted lipid DSPE-PEG-mal. The cells treated with targeted lipoplex express significantly more amounts of reporter gene than that of the non-targeted lipoplex-treated cells. The highest differences in expression (8–12-fold, p < 0.001) were at a cationic lipid/DNA charge ratio of 4:1 and 2:1. Fig. 3A also shows that Lipofectamine™, one of the most frequently used cationic lipid-based gene transfection reagent, has significantly less effect in transfecting DNA in MCF-7 cells; thereby, proving the utility of the DSPE-PEG-HP-containing targeted lipoplex to transfect MCF-7 cells. Fig. 3B shows the comparison between reporter gene expression in MCF-7 cells treated with targeted lipoplex in the absence or presence of either HP or DTG. With a cationic lipid/DNA charge ratio of 4:1 the difference in reporter gene expression is maximum (p < 0.001). With a 2:1 charge ratio, the difference in transgene expression is significant in the case of HP (p < 0.001) and is nearly significant for DTG (p = 0.008). The result proves that the uptake of targeted lipoplex is indeed mediated through sigma receptors. Fig. 3C shows a similar comparison in MCF-7 cells treated with non-targeted cationic lipoplexes in the presence or absence of either HP or DTG. The overall gene expression is severalfold lower compared with that obtained through targeted lipoplex. Simultaneously, there is no consistent evidence of targeted uptake and eventual gene expression in cells treated with non-targeted cationic lipoplex. The gene expression was nonspecific in all the charge ratios and remains uninhibited in the presence of either HP or DTG. Gene Transfection in CHO Cells—CHO, a non-human cell, is known to possess very low levels of sigma receptors (26Mei J. Pasternak G.W. Biochem. Pharmacol. 2001; 62: 349-355Crossref PubMed Scopus (112) Google Scholar). It is one of the most transfectable cell lines by cationic lipids. Fig. 3D shows the relative comparison between the reporter gene expression in CHO cells treated with targeted cationic lipoplex in the presence or absence of HP. Fig. 3E shows the relative comparison of gene expression in CHO cells treated with non-targeted cationic lipoplex in the presence or absence of HP. The gene expression is relatively high because of known nonspecific cell surface interaction with transfecting cationic lipids. However, the difference in gene expression in cells pretreated or untreated with HP is shown to be mostly insignificant, and no specific comparable trend is observed between cells treated with targeted and non-targeted lipoplexes. Hence, the sigma receptor has no role in targeted lipoplex-mediated gene transfection in CHO. Comparison of Sigma Receptor Levels in MCF-7 and CHO Cells—To correlate the difference in targeted transfection in MCF-7 cells compared with that obtained in CHO with the present levels of sigma receptors in respective cells, a Western blot analysis was undertaken for the cellular proteins of the two cell lines. Fig. 4 shows the Western blot analysis of MCF-7 and CHO for the detection of the sig
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