Novel cationic solid-lipid nanoparticles as non-viral ...

Downloaded By: [University of Torino] At: 13:24 11 May 2007

Journal of Drug Targeting, May 2007; 15(4): 295–301

Novel cationic solid-lipid nanoparticles as non-viral vectors for gene delivery MARIA LUISA BONDI’1, ANTONINA AZZOLINA2, EMANUELA FABIOLA CRAPARO3, NADIA LAMPIASI2, GIULIA CAPUANO3, GAETANO GIAMMONA3, & MELCHIORRE CERVELLO2 1

Istituto per lo Studio dei Materiali Nanostrutturati, Consiglio Nazionale delle Ricerche, Via Ugo La Malfa 153, 90146 Palermo, Italy, 2Istituto di Biomedicina e Immunologia Molecolare, Consiglio Nazionale delle Ricerche, Via Ugo La Malfa 153, 90146 Palermo, Italy, and 3Dipartimento di Chimica e Tecnologie Farmaceutiche, Universita` di Palermo, via Archirafi 32, 90123 Palermo, Italy

(Received 27 January 2007; revised 7 March 2007; accepted 7 March 2007)

Abstract In this paper, the suitability of novel cationic solid-lipid nanoparticles (SLN) as a nonviral transfection agent for gene delivery was investigated. SLN were produced by using the microemulsion method and Compritol ATO 888 as matrix lipid, dimethyldioctadecylammonium bromide as charge carrier and Pluronic F68 as surfactant. Obtained nanoparticles were approximately 120 nm in size and positively charged, with a zeta potential value equal to þ45 mV in twice-distilled water. Cationic SLN were able to form stable complexes with DNA and to protect DNA against DNase I digestion. The SLN– DNA complexes were characterized by mean diameter and zeta potential measurements. In vitro studies on human liver cancer cells demonstrated a very low degree of toxicity of both SLN and SLN – DNA complexes. Further, SLN – DNA complexes were able to promote transfection of liver cancer cells. These data suggest that our cationic SLN may be potentially useful for gene therapy.

Keywords: Gene therapy, cationic solid-lipid nanoparticles, non-viral DNA vectors, cytotoxicity, cell transfection, liver cancer

Introduction Gene therapy is an area of considerable current interest that is fast becoming a reality. Despite that naked DNA was used successfully when injected directly into the tumor (Shi et al. 2002; Walther et al. 2002) or as DNA vaccines (Conry et al. 1998; Hanke et al. 2002), it is highly prone to tissue clearance and totally inefficient after intravenous administration (Kawabata et al. 1995). Since the effectiveness of a gene therapy is determined mainly by a vector system (Nabel 1999; Benns and Kim 2000) research has been focused on designing efficient vectors (Peng and Vile. 1999; El-Aneed 2004). Thanks to these systems, genetic material such as DNA, RNA and oligonucleotides have been used as molecular medicine and are delivered to specific cell types with the aim to either

inhibit some undesiderable gene expression or express therapeutic proteins (Davis 1997; Anderson 1998). A variety of gene transfer systems are currently employed to insert therapeutics genes into somatic cells and are mainly divided into viral vectors and nonviral vectors (El-Aneed 2004). Viral vectors are biological systems derived from naturally evolved viruses capable of transferring genetic materials into the host cells. While viral vectors provide efficient gene delivery, they have serious drawbacks in terms of potential pathogenicity because of the possible viral recombination, and the risk of an immune or inflammatory response (ElAneed 2004). These limitations have encouraged researchers to increasingly focus on non-viral vectors as an alternative to viral vectors.

Correspondence: M. L. Bondi’, Istituto per lo Studio dei Materiali Nanostrutturati, Consiglio Nazionale delle Ricerche, Via Ugo La Malfa 153, 90146 Palermo, Italy. Tel: 39 91 6809367. Fax: 39 91 6809247. E-mail: [email protected] ISSN 1061-186X print/ISSN 1029-2330 online q 2007 Informa UK Ltd. DOI: 10.1080/10611860701324698


296 M. L. Bondi’ et al. Non-viral vectors are generally cationic and interact with the negatively charged DNA through electrostatic interactions. They include cationic polymers (Itaka et al. 2003; Licciardi et al. 2006); cationic peptides (Weijun et al. 2004) and cationic liposomes (Tabatt et al. 2004). Although non-viral vectors are less efficient than viral ones, they have the advantages of safety, simplicity of preparation and high gene encapsulation capability. In this field, minimal attention has been paid to the use of solid-lipid nanoparticles (SLN) as DNA carriers, although these may offer a number of technological advantages. These include excellent storage stability, a relatively easy production without the use of any organic solvent, the possibility of steam sterilization and lyophilization, and large scale production (Schwarz and Mehnert 1995; Schwarz and Mehnert 1997; Mehnert and Mader 2001). Moreover, SLN are obtained by using physiologically well-tolerated ingredients already approved for pharmaceutical applications in humans (Wissing et al. 2004) and show low toxicity when injected intravenously (Yang et al. 1999). In addition, an advantage of SLN is that the charge of the particles can be modulated via the composition, thus allowing binding of oppositely charged molecules via electrostatic interactions. SLN can be produced in nano-scale size, in which the particles are sufficiently small to traverse the microvascular system and prevent macrophage uptake and are therefore particularly suitable for systemic delivery. Recently, lipid nanoparticles bearing cationic groups have been shown to efficiently bind and transfect plasmid DNA into mammalian cells in vitro (Olbrich et al. 2001; Pedersen et al. 2006). Different reporter genes have been used to monitor gene expression in vitro. The expression genes coding for different enzymes, including b-galactosidase, can be estimated by measuring the corrisponding activated substrate. Our current research interests deal with possible strategies able to treat hepatocellular carcinoma (HCC). For most patients with advanced HCC treatment options are limited. Novel therapeutic strategies such as gene therapy are therefore urgently required. Pre-clinical evidence and early clinical trials strongly suggest that there is a place for gene therapy in liver cancer. The aim of the present study was to develop a SLN vector containing a cationic modifier and to evaluate its potential as a gene transfection agent. In the first step, we have prepared and characterized cationic SLN in terms of mean size, zeta potential and in vitro cytotoxicity and we have evaluated their ability to complex plasmid DNA as a function of cationic SLN:DNA weight ratio. Subsequently, we have investigated the ability of cationic SLN –DNA complexes to protect DNA from DNase I digestion and to transfect DNA into human liver cancer cells.

Materials and methods Materials Compritol ATO 888 (mixture of mono-, di- and triglycerides of behenic acid) was a gift from Gattefosse´ (D-Weil am Rhein, Germany). Pluronic F68 and the dimethyldioctadecylammonium bromide (DDAB) were purchased from Sigma-Aldrich (Milan, Italy). Amplification and purification of plasmid DNA (pCMVb-gal) Plasmid DNA encoding the b-galactosidase gene under the control of the human cytomegalovirus (CMV) was used in this study as the reporter gene. The plasmid pCMV-b-gal was transformed into Escherichia coli XL-1 blue bacterial strain. The transformed cells were grown in LB broth supplemented with 50 mg/ml ampicillin. The plasmid DNA was purified using a GenElute Endotoxin-free Plasmid Midiprep Kit (Sigma, Milan, Italy) to remove the bacterial endotoxins, and the purified plasmid was diluted in sterile water. The purity was confirmed by 0.8% agarose gel electrophoresis using a Tris – borate – EDTA (TBE) buffer system, followed by ethidium bromide staining and DNA concentration was measured by UV absorption at 260 nm. Preparation of cationic SLN Cationic SLN were prepared from a warm oil-in-water (o/w) microemulsion by using Compritol ATO 888 and DDAB as lipid matrix. Briefly, 0.273 mmole of compritol were heated to 108C above its melting point and mixed with a 2.5 ml of a hot aqueous solution of Pluronic F68 (0.0158 mmole) and DDAB (1.45 mmole) to form a clear microemulsion, under mechanical stirring. Then, cationic nanoparticles were obtained by dispersing the warm o/w microemulsion in cold water (2– 38C) (organic:aqueous volume ratio equal to 1:10) under mechanical stirring at 1000 rpm. The obtained cationic nanoparticles were purified by dialysis using a Visking Tubing Dialysis 18/3200 (with a molecular weight cut-off of 12,000 – 14,000 D). Then, cationic nanoparticles were freeze-dried by using a Modulyo freeze-dryer (Labconco Corporation, Missouri, USA) and stored in the dark and at room temperature for further characterization by photon correlation spectroscopy (PCS) and zeta potential measurements. Preparation of SLN – DNA complexes and DNA retardation assay Cationic SLN were dispersed in twice-distilled water, filtered through a 0.2 mm nylon filter (Millipore, Milan, Italy) and lyophilized. Subsequently, they were weighed and dispersed in twice-distilled water at a


Novel cationic solid-lipid nanoparticles concentration of 1 mg/ml by using a water bath sonication for 15 min (T310, Elma, Germany). The SLN – DNA complexes were prepared by mixing 200 ng DNA in distilled H2O with the desired amount of complexing agent in a final volume of 10 ml. After a 30 min incubation at room temperature, DNA binding was studied by assaying for agarose gel retardation. Samples were electrophoresed through a 0.8% agarose gel using a TBE buffer system. DNA was visualized using ethidium bromide staining. To evaluate the stability of cationic SLN –DNA complexes as a function of incubation time, a gel retardation assay was performed on samples obtained at cationic SLN:DNA weight ratios of 50:1 and 100:1, after 30, 60 and 120 min incubation times at room temperature. Size and zeta potential measurements Particles and complexes size were analyzed by PCS using a Zetasizer Nano ZS (Malvern Instrument, Herrenberg, Germany) which utilizes non-invasive back-scattering (NIBS) technique. PCS gives information about the mean diameter of the bulk population (so-called z-average) and the width of distribution via the polydispersity index (PI). Samples were appropriately diluted with filtered (0.2 mm) twice-distilled water and the readings were carried at a 1738 angle in respect to the incident beam. The reported values were the average of three measurements. The surface charge was determined using the same equipment. Zeta potential values were measured using principles of Laser Doppler Velocimetry and Phase Analysis Light Scattering (M3-PALS technique). Samples were dispersed in filtered (0.2 mm) twicedistilled water and analyzed in triplicate. DNase I degradation assay To evaluate the sensitivity of the SLN – DNA complexes to DNase I digestion, preformed complex at 200:1 weight ratio of cationic SLN:DNA (containing 2 mg of DNA) and naked DNA (2 mg) were mixed with 2 U of DNase I in 100 ml total volume containing 25 mM Tris –HCl pH 7.5 and 5 mM MgCl2. After 30 min at 378C, the samples were extracted with equal volumes of phenol, phenol/chloroform and DNA in the aqueous phase was then precipitated by adding sodium acetate and ethanol. DNA was suspended again in 20 ml of twice-distilled water, and 10 ml were electrophoresed through a 0.8% agarose gel to examine the DNA size. Cell culture The human hepatoma HuH-6 cell line was kindly provided by Professor Massimo Levrero (Laboratory

297

of Gene Expression, Fondazione Andrea Cesalpino, University of Rome “La Sapienza”, Rome, Italy) and cultured in Minimum Essential Medium Eagle (MEM) (Sigma, Milan, Italy) supplemented with 10% heat-inactivated fetal calf serum (FCS) (Gibco, Milan, Italy), 2 mM L -glutamine, 1 mM sodium pyruvate, 100 U/ml penicillin and 100 mg/ml streptomycin (all reagents were from Sigma) in a humidified atmosphere at 378C in 5% CO2. Cells having a narrow range of passage number were used for all experiments. Cytotoxicity Cytotoxicity was assessed by the MTS assay and by using the CellTiter Aqueous OneSolution kit (Promega Corporation, Madison, WI, USA) according to the manufacturer’s instructions. Briefly, cells (5 £ 103/well) in complete medium were distributed into each well of 96-well microtiter plates and then incubated overnight. At time 0, the medium was replaced with fresh complete medium either with SLN or SLN – DNA complexes. SLN – DNA complexes were prepared by adding the desired amount of particle suspension to plasmid DNA in water to obtain a cationic SLN:DNA weight ratio ranged from 5:1 to 200:1. After 30 min at room temperature, the complexes were diluted with one volume of 2 £ concentrated RPMI complete medium and added to the cells. Cells were cultured for 48 h and at the end of treatment with various concentrations of the reagents, 15 ml of a commercial solution (Promega Corporation, Madison, WI, USA) containing 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulphophenyl)-2H-tetrazolium (MTS) and phenazine ethosulfate were added to each well. The plates were incubated for 1 h in a humidified atmosphere at 378C in 5% CO2. The bioreduction of the MTS dye was assessed by measuring the absorbance of each well at 490 nm. Cytotoxicity was expressed as a percentage of the absorbance measured in the control cells (100% viability). Values were expressed as means ^ SD of three separate experiments, each in triplicate. Transfection procedure and b-galactosidase measurements To assess b-galactosidase activity, 2.5 £ 104/well of HuH-6 cells were seeded on 24-well plates and transfected by using FuGENE 6 transfection reagent (Roche, Milan, Italy) and SLN – DNA complexes obtained with a cationic SLN:DNA weight ratio ranged from 5:1 to 200:1. Complexes were performed by mixing SLN and DNA in twice-distilled water at room temperature for 30 min. Samples were diluted in 2 £ MEM and 25 ml of each suspension were added to each well. After 4 h, 100 ml/ml of FCS was added to each well (final concentration 10%). After 48 h cells were washed with


298 M. L. Bondi’ et al. PBS and lysed for 20 min on ice with 100 ml of RLB buffer (Promega). After a cycle of freezing and thawing, an aliquot of 10 ml of each sample was withdrawn and used for determination of protein content. Samples were diluted up to 250 ml with RLB and then 250 ml of 2 £ assay buffer (consisting of 20 mM KCl, 2 mM MgSO4, 100 mM b-mercapthoethanol, 1.33 mg/ml o-nitrophenyl-b-D -galactopyranoside (ONPG), 400 mM sodium phosphate buffer) at pH 7.0 were added. After 30 min absorbance at 405 nm was measured. These experiments were also performed in the absence of FCS and no differences in the trasfection efficiency of cationic SLN –DNA complexes were evidenced. The protein content of the transfected cells was measured using the Bio-Rad protein assay kit (BioRad Laboratories, Milan, Italy). Transfection experiments were performed in triplicate at least two times, and b-galactosidase activity, expressed in arbitrary units, was normalized for total cell protein content. Statistical analyses Comparison between groups were performed with Student’s test and a P value , 0.05 was considered significant.

amounts of cationic SLN in such way to obtain cationic SLN:DNA weight ratios ranging from 10:1 to 200:1. The efficiency of DNA complexation by cationic SLN after 30 min of incubation was evaluated by the amount of cationic SLN required to retard the migration of plasmid DNA toward the cathode during agarose gel electrophoresis (Figure 1A). As can be seen in Figure 1, cationic SLN were able to immobilize DNA at a cationic SLN:DNA weight ratio around 100:1. At higher concentrations, SLN were even able to prevent intercalation of ethidium bromide in DNA (Figure 1A, lane 7). We assayed also whether incubation time influences the efficiency of DNA complexation by cationic SLN. In particular, on SLN –DNA complexes prepared with cationic SLN:DNA weight ratios equal to 50:1 and 100:1 (the latter being the lowest weight ratio that gives the retardation of DNA migration) an agarose gel electrophoresis was carried out after 30, 60 and 120 min of incubation in twice-distilled water (Figure 1B). The results obtained show that prolonging the incubation time up to 120 min, the efficiency of DNA complexation only slightly improves in the case of the SLN:DNA ratio equal to 50:1, whereas it does not influence the complexation at a SLN:DNA weight ratio equal to 100:1. The physical properties of cationic SLN –DNA complexes were determined by PCS and zeta potential

Results and discussion Preparation and characterization of cationic SLN A novel cationic SLN formulation was produced from a warm o/w microemulsion by using as lipid materials compritol 888 ATO and the cationic lipid DDAB, and as surfactant pluronic F68. Obtained particles were characterized by mean diameter and zeta potential measurements, which confirmed respectively their nanometric size and positive surface charge. In particular, the average particle size was 125 nm (PI ¼ 0.252), that is sufficiently small to make these nanoparticles particularly suitable for systemic administration. The surface charge value was highly positive (þ 45 mV), demonstrating the incorporation of DDAB successfully onto the nanoparticle surface. The cationic SLN stored in the dark and at room temperature showed excellent storage stability, since the particle diameter and PI changed only by a few nanometers during the storage time of 180 days (data not shown). Interaction between cationic SLN and plasmid DNA The interaction between cationic SLN and DNA was investigated by retardation of the DNA electrophoretic mobility. The complexes were formed in twice-distilled water by mixing fixed amount of DNA with increasing

Figure 1. Evaluation of the capacity of SLN to complex plasmid DNA. (A) Increasing amounts of SLN were mixed with a constant amount of plasmid DNA (200 ng) in twice-distilled water for 30 min. The complexes were then electrophoresed through a 0.8% agarose gel. Lanes from left: M, molecular weight marker, DNA only, SLN:DNA mixtures with w/w ratios of 10:1, 25:1, 50:1, 100:1 and 200:1. (B) Cationic SLN–DNA complexes with a weight ratio of 50:1 and 100:1 were incubated for 30, 60 and 120 min and then electrophoresed through a 0.8% agarose gel. (C) Naked plasmid DNA.


Novel cationic solid-lipid nanoparticles Table I. Mean diameter and PI of cationic SNL–DNA complexes at different SLN:DNA weight ratios, after 30 min of incubation in twice-distilled water. SLN/DNA weight ratio (w/w) 20:1 40:1 50:1 80:1 100:1 150:1 200:1

Mean diameter (nm) (^SD)

PI

146 (25) 187 (32) 283 (28) 311 (29) 389 (31) 461 (23) 588 (33)

0.292 0.323 0.358 0.383 0.414 0.434 0.565

measurements. Table I shows values of mean diameter and PI in twice-distilled water of the cationic SLN – DNA complexes obtained with cationic SLN:DNA weight ratios ranging between 20:1 and 200:1. In particular, the mean size of SLN – DNA complexes rises from 146 to 588 nm as the SLN:DNA weight ratio increases from 20:1 to 200:1. This fact reasonably could be explained considering that one moiety of DNA can interact with several particles to form a complex thanks to the opposite charge between DNA and SLN. These size values should be suitable for uptake by endocytotic processes or direct fusion with the cell membrane (Olbrich et al. 2001). The trend of zeta potential measurements of complexes in twice-distilled water as a function of cationic SLN:DNA weight ratio is illustrated in Figure 2. It is shown that by increasing the amount of cationic SLN used for the complex formation, zeta potential of the obtained complexes values increase, starting from 2 42 mV for a dispersion of naked DNA, and become positive for a cationic SLN:DNA weight ratio equal to 100:1.

299

DNase I degradation assay The degradation of DNA by nucleases could dissuade the administration of genetic material in vivo; for this reason an efficient gene vector should be able to stabilize DNA and to prevent its degradation. To detect the capability of cationic SLN to protect DNA from enzymatic degradation, the DNase I was selected as a model enzyme for carrying out a degradation assay; moreover the complex obtained by using a cationic SLN:DNA weight ratio equal to 200:1 was tested. Figure 3 shows the protection effect of cationic SLN on DNA in the complexed form compared with naked DNA, both incubated for 30 min in the absence and in the presence of DNase I at 378C. After this time, the plasmid DNA was extracted from each bath and then loaded onto a 0.8% agarose gel to examine its integrity (see experimental section). We found that while naked plasmid was completely digested after incubation with DNase I, plasmid derived from the complex with cationic SLN was not degraded. Effect of cationic SLN and SLN– DNA complexes on cell viability Cationic SLN and SLN –DNA complexes were also characterized with regard to their effect on in vitro viability of human liver cancer cells HuH-6 by MTS assay. Cells were incubated for 48 h in the presence of different concentrations of cationic SLN (ranging from 0 to 200mg/ml) and SLN –DNA complexes with a weight ratio up to 200:1. As reported in Figure 4, the dose-response study demonstrated that cationic SLN and SLN –DNA complexes showed very low degrees of cytotoxicity on HuH-6 cells.

Figure 2. Zeta potential values of cationic SLN– DNA complexes. Increasing amounts of SLN were mixed with a constant amount of plasmid DNA (200 ng) in twice-distilled water for 30 min. The complexes were then analyzed using principles of M3-PALS technique. Data are the mean ^ SD of three separate experiments, each of which was performed in triplicate.


300 M. L. Bondi’ et al.

Figure 3. Agarose gel electrophoresis of DNA after DNase digestion. Naked DNA and cationic SLN–DNA complex at a cationic SLN:DNA weight ratio equal to 200:1 were incubated in the absence and in the presence of DNase I for 30 min at 378C. Samples were then subjected to extraction and precipitation, loaded onto a 0.8% agarose gel and electrophoresed to examine the integrity of the plasmid DNA.

Evaluation of the transfection efficiency The ability of cationic SLN to transfect the pCMVbreporter gene plasmid at a fixed concentration of DNA into HuH-6 cells was tested in the absence and in the presence of serum. As control reagent, we chose the widely used FuGeneTM 6, a commercially available transfecting agent. As shown in Figure 5A, in the absence of serum, cationic SLN were able to promote DNA transfection in a dose-dependent manner, while naked DNA did not produce any detectable of b-galactosidase activity. The transfection efficiency of SLN:DNA complexes was comparable to that obtained using by FuGene 6 reagent. To mimic the systemic administration conditions, transfection was performed in a cell culture medium supplemented with serum. In the presence of 10% foetal bovine serum results were similar to those obtained in the absence of serum (Figure 5B).

Figure 5. Transfection efficacy of SLN–DNA complexes on HuH-6 cell line. Cells were incubated with cationic SLN–DNA complexes at different cationic SLN:DNA weight ratios in the absence (A) or presence (B) of FCS. After 48 h, cells were lysed and b-galactosidase activity was measured. Data are normalized for total protein content and transfection efficacy is expressed as arbitrary units. Data are the mean ^ SD of three separate experiments, each of which was performed in triplicate. *P , 0.05; **P , 0.005, vs. naked DNA.

Conclusion

Figure 4. In vitro cytotoxicity of cationic SLN and SLN– DNA complexes on HuH-6 cells. Cells were incubated in the presence of cationic SLN– DNA complexes at different cationic SLN:DNA weight ratios, or equivalent amount of SLN, for 48 h and then cell viability was assessed by MTS assay. Data are expressed as percentage on untreated cells (100% viability) and are the mean ^ SD of three separate experiments, each of which was performed in triplicate. *P ¼ ns (not significant), vs. control.

Development of safe and efficient non-viral systems for gene delivery is a major challenge in the field of gene therapy. In this study, with the aim to obtain cationic nanoparticles potentially useful as plasmid transfection vector for gene delivery, we prepared cationic SLN based on Compritol ATO 888 and DDAB and characterized them regarding particle size, surface charge, DNA binding, cytotoxicity and transfection efficiency. Obtained cationic SLN were successfully produced by the microemulsion method and were able to bind efficiently DNA to achieve a shift in its electrophoretic mobility. Also the ethidium bromide exclusion test clearly demonstrates that we were successful in the complexation of the DNA with cationic SLN. In particular, complexes obtained with a cationic SLN:DNA weight ratio equal to 100:1 and 200:1 were able to immobilize the DNA and possess a mean size suitable for the intravenous administration. Zeta


Novel cationic solid-lipid nanoparticles potential measurements also confirmed the interaction between polyanionic DNA and cationic SLN. Moreover, complexes were able to protect DNA from DNase I digestion. An important aspect of transfection agents, especially for non-viral systems, is the efficiency/toxicity ratio. We showed that when cationic SLN:DNA weight equivalents in the range between 25 and 200 are used, DNA is transfected in HuH-6 cells, and this good transfection efficiency was associated with very low cytotoxicity degree. These data suggest that cationic SLN here described may be safe and may efficiently delivery complexed DNA, supporting their potential use for in vivo applications as non-viral transfection agents.

References Anderson WF. 1998. Human gene therapy. Nature 392:25–30. Benns JM, Kim SW. 2000. Tailoring new gene delivery designs for specific targets. J Drug Target 8:1–12. Conry RM, White SA, Fultz PN, Khazaeli MB, Strong TV, Allen KO, Barlow DL, Moore SE, Coan PN, Davis I, Curiel DT, Lo Buglio AF. 1998. Polynucleotide immunization of nonhuman primates against carcinoembryonic antigen. Clin Cancer Res 4:2903–2912. Davis SS. 1997. Biomedical applications of nanotechnologyimplications for drug targeting and gene therapy. Trends Biotechnol 15:217–224. El-Aneed A. 2004. An overview of current delivery systems in cancer gene therapy. J Control Rel 94:1–14. Hanke P, Serwe M, Dombrowski F, Sauerbruch T, Caselmann WH. 2002. DNA vaccination with AFP-encoding plasmid DNA prevents growth of subcutaneous AFP-expressing tumors and does not interfere with liver regeneration in mice. Cancer Gene Ther 9:346–355. Itaka K, Yamauchi K, Harada A, Nakamura K, Kawaguchi H, Kataoka K. 2003. Polyion complex micelles from plasmid DNA and poly(ethylene glycol)-poly(l-lysine) block copolymer as serum-tolerable polyplex system: Physicochemical properties of micelles relevant to gene transfection efficiency. Biomaterials 24:4495– 4506. Kawabata K, Takakura Y, Hashida M. 1995. The fate of plasmid DNA after intravenous injection in mice: Involvement of

301

scavenger receptors in its hepatic uptake. Pharm Res 12:825–830. Licciardi M, Campisi M, Cavallaro G, Cervello M, Azzolina A, Giammona G. 2006. Synthesis and characterization of polyaminoacidic polycations for gene delivery. Biomaterials 27:2066–2075. Mehnert W, Mader K. 2001. Solid lipid nanoparticles: Production, characterization and applications. Adv Drug Deliv Rev 47:165–196. Nabel GJ. 1999. Development of optimized vectors for gene therapy. Proc Natl Acad Sci USA 96:324–326. Olbrich C, Bakowsky U, Lehr CM, Muller RH, Kneuer C. 2001. Cationic solid lipid nanoparticles can efficiently bind and transfect plasmid DNA. J Control Rel 77:345–355. Pedersen N, Hansen S, Heydenreich AV, Kristensen HG, Poulsen HS. 2006. Solid lipid nanoparticles can effectively bind DNA, streptavidin and biotinylated ligands. Eur J Pharm Biopharm 62:155–162. Peng KW, Vile R. 1999. Vector development for cancer gene therapy. Tumor Target 4:3–11. Schwarz C, Mehnert W. 1995. Sterilization of drug-free and tetracaine-loaded solid lipid nanoparticles (SLN), Proceeding of the First World Meeting APGI/APV, Budapest p 485 –486. Schwarz C, Mehnert W. 1997. Freeze-drying of drug-free and drugloaded solid lipid nanoparticles (SLN). Int J Pharm 157:171–179. Shi F, Rakhmilevich AL, Heise CP, Oshikawa K, Sondel PM, Yang NS, Mahvi MD. 2002. Intratumoral injection of interleukin-12 plasmid DNA, either naked or in complex with cationic lipid, results in similar tumor regression in a murine model. Mol Cancer Ther 1:949–957. Tabatt K, Sameti M, Olbrich C, Muller RH, Lehr CM. 2004. Effect of cationic lipid and matrix lipid composition on solid lipid nanoparticles-mediated gene transfer. Eur J Pharm Biopharm 57:155–162. Walther W, Stein U, Fichtner I, Voss C, Schmidt T, Schleef M, Nellessen T, Schlag PM. 2002. Intratumoral low-volume jetinjection for efficient nonviral gene transfer. Mol Biotechnol 21:105–115. Weijun L, François N, Jr, Francis SC. 2004. GALA: A designed synthetic pHresponsive amphipathic peptide with applications in drug and gene delivery. Adv Drug Del Rev 56:967–985. Wissing SA, Kayser O, Muller RH. 2004. Solid lipid nanoparticles for parenteral drug delivery. Adv Drug Del Rev 56:1257– 1272. Yang SC, Lu LF, Cai Y, Zhu JB, Liang BW, Yang CZ. 1999. Body distribution in mice of intravenously injected camptothecin solid lipid nanoparticles and targeting effect on brain. J Control Rel 59:299–307.