DOI: 10.1002/cbic.201500208
Full Papers
Folic-Acid-Targeted Self-Assembling Supramolecular Carrier for Gene Delivery Rongqiang Liao,[a] Shouhui Yi,[b] Manshuo Liu,[a] Wenling Jin,[a] and Bo Yang*[a] A targeting gene carrier for cancer-specific delivery was successfully developed through a “multilayer bricks-mortar” strategy. The gene carrier was composed of adamantane-functionalized folic acid (FA-AD), an adamantane-functionalized poly(ethylene glycol) derivative (PEG-AD), and b-cyclodextrin-grafted low-molecular-weight branched polyethylenimine (PEI-CD). Carriers produced by two different self-assembly schemes, involving either precomplexation of the PEI-CD with the FA-AD and PEG-AD before pDNA condensation (Method A) or pDNA condensation with the PEI-CD prior to addition of the FA-AD
and PEG-AD to engage host–guest complexation (Method B) were investigated for their ability to compact pDNA into nanoparticles. Cell viability studies show that the material produced by the Method A assembly scheme has lower cytotoxicity than branched PEI 25 kDa (PEI-25KD) and that the transfection efficiency is maintained. These findings suggest that the gene carrier, based on multivalent host–guest interactions, could be an effective, targeted, and low-toxicity carrier for delivering nucleic acid to target cells.
Introduction In gene carriers, cationic polymers possess several advantages: they have, for example, high transfection capability and low toxicity, they are non-immunogenic, and their chemical and structural properties can be tightly controlled. This allows carriers that are suitable for mass production to be designed.[1] Cationic polymers can spontaneously interact with nucleic acids to form polyplexes through electrostatic interactions. These polyplexes are usually 50–200 nm nanoparticles with positive surface charge; they are thought to interact electrostatically with negatively charged cell membranes to promote cellular uptake through nonspecific endocytosis.[2] Of those cationic polymers, polyethylenimine (PEI) is a promising delivery reagent, primarily because of its excellent transfection efficiency in a wide range of cell types.[3] By virtue of its abundant primary amine groups, PEI readily forms polyplexes with negatively charged nucleic acids and subsequently buffers the endosomal environment, facilitating the release of nucleic acid in the cytosol.[4] However, it should be noted that the transfection efficiency and cytotoxicity of PEI are dependent on its molecular weight. In general, PEI with higher molecular weight has a higher transfection efficiency, but also higher cytotoxicity. The primary reason for the cytotoxicity is that the positive charge on a polyplex can also interact with a wide variety of negatively charged components in vivo.[5] Consequent[a] R. Liao, M. Liu, W. Jin, Prof. B. Yang Faculty of Life Science and Technology Kunming University of Science and Technology Kunming 650500 (P. R. China) E-mail: [email protected] [email protected] [b] S. Yi Department of Oncology, Chongqing Cancer Hospital Chongqing 400011 (P. R. China)
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ly, shielding agents have to be introduced onto polyplex particle surfaces in order to prevent such nonspecific interactions. One common approach to shielding excessive positive charge is to attach poly(ethylene glycol) (PEG) chains onto the polyplex particle surface. This strategy, often termed PEGylation, can effectively improve serum stability and reduce cytotoxicity.[6] In addition to modification of cationic polymers, gene carriers—a variety of different target-specific ligands, including various transfer proteins and small-molecule compounds such as folic acid (FA)—have also been explored with the goal of enhancing target-specific gene delivery.[7] In recent years, FA has been widely utilized as a promising targeting ligand because folate receptors (FRs) are overexpressed in many human tumor cells, including malignancies of the colon, rectum, ovary, kidney, breast, and lung, but have little expression in normal tissues.[8] Similarly, b-cyclodextrin (CD) is widely used for gene delivery because of its derivatives’ excellent properties such as low toxicity, high biodegradability, stability, and biocompatibility and facile chemical modification.[9] Additionally, CD has been used as a convenient building block for the construction of new functional and nanostructured materials. A variety of CD-based systems such as CD polymers, CD polyrotaxanes, and CD dendrimers are promising materials for nonviral vector development.[10] Recently, Davis reported a diverse class of CD oligomers coupled through cationic linkers, and successfully used them as vectors for siRNA delivery in a clinical trial for treatment of melanoma in humans.[11] CD can host various poorly soluble molecules in its hydrophobic cavity, with high size- and shape-selectivity both in aqueous solution and in the solid state.[12] With some molecules, this association can be quite strong. For example, the association constants (Ka) for adaman-
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Full Papers tane (AD) derivatives and CD are of the order of 104– 105.[13] This noncovalent self-assembly method has many advantages, such as the lack of need for special reaction equipment, mild reaction conditions, simplicity of operation, environmental friendliness, and easy batch preparation. The AD guest molecule can be PEGylated with PEG to improve serum stability and to reduce cytotoxicity, and can be vectorized with FA to target folate receptors on the tumor cell surface. Introduction of a gene vector based on CD onto such adamantane-functionalized molecules can improve the transfection efficiency significantly. Here we have employed this supramolecular strategy to achieve self-assembly of a nucleic acid delivery vector from three different molecular building blocks: namely, 1) b-cyclodextrin-grafted low-molecular-weight branched polyethylenimine (PEI-CD), 2) adamantane-functionalized folate (FA-AD), and 3) an adamantane-functionalized poly(ethylene gly- Figure 2. 1H NMR spectrum of PEI-CD. col) derivative (PEG-AD). It was anticipated that plasmid DNA (pDNA) compaction might be achievable through multivalent electrostatic interactions between the PEIof the gene delivery systems. PEI-CD was synthesized by nucleCD and the pDNA, and that the CD units in the PEI-CD could ophilic substitution of the tosyloxy group in b-CD-OTs by an host the AD groups of the FA-AD and PEG-AD through hydroamino group of PEI in the presence of potassium carbonate as phobic interactions. This design could enable the compaction the catalyst. As shown in Figure 2, the CD protons displayed of pDNA into stable nanometer-sized particles that can then chemical shifts at d = 4.7 ppm and 3.0–3.8 ppm, distinct from be targeted and internalized within tumor cells through folate those of the PEI protons (d = 2.0–2.7 ppm). This result indicated receptor recognition at the tumor cell surface (Figure 1). that the PEI had been linked to the CD. In the PEI-CD molecule there were about six CD units grafted on a branched PEI backbone according to the 1H NMR spectrum. The successful preparation of PEI-CD·PEG-AD·FA-AD complexes was also clearly demonstrated by the 1H NMR spectrum shown in Figure 3. AD was insoluble in H2O, but there were AD proton peaks in the system in D2O. This result indicated that the AD was bound in the CD cavity. To further demonstrate the presence of the AD in the CD cavity, the chemical shifts (d) of the complexes were compared (Table 1). After inclusion complexation with AD, the H3 proton of CD had shifted by 0.110 ppm, the H5 proton of CD by 0.090 ppm, and the protons of AD by ca. 0.400 ppm. These results thus provided further evidence that the AD was included in the CD cavity.
Table 1. The chemical shifts (d) of AD, PEI-CD, and the AD·PEI-CD complex.
Figure 1. Conceptual diagram of polycation/pDNA complexation.
Results and Discussion Preparation of PEI-CD, PEG-AD, FA-AD, and complexes In this work, we constructed the folate-targeted gene vector. The PEI-CD·PEG-AD·FA-AD complexes to be used as the final gene carriers in this study could be simply obtained by a “bricks and mortar” strategy. The self-assembly process should facilitate future quality control and manufacturing procedures ChemBioChem 2015, 16, 1622 – 1628
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H1 of CD H2 of CD H3 of CD H4 of CD H5 of CD H6 of CD H1 of AD H2 of AD H3 of AD
d dd dd dd m dd m d m
AD
PEI-CD
Complex
– – – – – – 1.645 1.805 1.925
4.710 3.275 3.590 3.225 3.515 3.545 – – –
4.725 3.305 3.700 3.335 3.605 3.595 1.125 1.455 1.565
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Full Papers Zeta potential is an indicator of surface charge on a polycation/pDNA nanoparticle, and a positive surface charge on a nanoparticle could electrostatically interact with a negatively charged cellular membrane. As indicated in Figure 5 B, the zeta potentials of Method A polyplexes have very low values, in the 0.5–2.5 mV range, regardless of N/P ratio, whereas the surface charges of Method B polyplexes increased from 0.8 to 6.9 mV with increasing N/P ratio. This might be caused by the neutral FA-AD and PEG-AD shielding the surface charge of PEI-CD·pDNA polyplexes for Method A. Figure 6 shows representative AFM images of the Method A and Method B polyplexes at an N/P ratio of 20. The AFM images distinctly revealed that most of the tight polyplexes produced by either of the two methods had average diameters of 200 nm or
Figure 3. 1H NMR spectrum of PEI-CD·PEG-AD·FA-ADa.
Biophysical characterization of the polyplexes obtained by both methods A successful gene delivery system requires that the nucleic acid be compacted by the polycation into nanoparticles small enough to facilitate cellular uptake.[14] In this work, the ability of PEI-CD·PEG-AD·FA-AD to condense pDNA into particulate structures was confirmed by agarose gel electrophoresis, measurement of zeta potentials, and atomic force microscopy (AFM). Two different complexation methods were used to evaluate the relative capacities of PEI-CD·PEG-AD·FA-AD host– guest polymer assemblies for pDNA condensation. In Method A, the pDNA was first complexed with the PEI-CD, followed by addition of the PEG-AD and FA-AD. In Method B, PEG-AD and FA-AD were preassociated with PEI-CD before addition to the pDNA solution. Gel-shift assays of the pDNA polyplexes with PEI-CD·PEGAD·FA-AD assemblies indicated that the two methods of formulation gave rise to slightly different pDNA complexation abilities. As shown in Figure 4, the polyplexes formed by Method A were able to inhibit DNA migration at an N/P ratio of 3. In the case of the polyplexes formed by Method B, slightly reduced pDNA complexation capability was observed, with an N/P ratio of 10 required to inhibit pDNA mobility completely. Due to partial shielding of surface positive charge and steric hindrance by the premixed FA-AD and PEG-AD towards PEICD, larger quantities of Method B PEI-CD·PEG-AD·FA-AD were generally necessary to achieve pDNA complexation than in Method A. Condensation of pDNA into nanoparticles in the size range of 50–200 nm was critical not only for in vivo distribution, but also for efficient cellular uptake by endocytosis through clathrin-coated pit mechanisms in cultured cells.[15] The effect of N/P ratio on particle size of complexes formed by both methods was investigated. As shown in Figure 5 A, the particle sizes of the two complexes decreased with increasing N/P ratio. Both methods could form nanoparticles of < 200 nm in average diameter at an N/P ratio of 30. However, the particle sizes of the Method B polyplexes were slightly larger than those of the Method A polyplexes. ChemBioChem 2015, 16, 1622 – 1628
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Figure 4. Agarose gel electrophoresis retardation of pDNA migration by polyplexes produced by Method A or Method B at various N/P ratios.
Figure 5. A) Particle size and B) zeta potential of polyplexes formed by both Methods A (~) and B (&).
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Full Papers sity. Meanwhile, the cytotoxicities of Method A polyplexes and Method B polyplexes showed no great difference. The main reason was also the introduction of PEG-AD and FA-AD.
Transfection efficiencies of the polyplexes produced by the two methods Figure 6. AFM images of A) pDNA, and of polyplexes produced by B) Method A, and C) Method B (N/P ratio of 20).
less; this was consistent with the results of the particle size measurement. In vitro cytotoxicity Gene carrier cytotoxicity has a great influence on transfection efficiency for pDNA delivery. In vitro toxicity study is essential to confirm the safety of any cationic polymers used for gene delivery. The primary reason for cytotoxicity is that the positive charge of a polyplex can also interact with a wide variety of negatively charged components in vivo. The presence of primary, secondary, and tertiary amines, which are usually charged under physiological conditions, is believed to induce gene vector cytotoxicity. Figure 7 shows cell viability as a function of cationic polymer concentration, determined by MTT assay in KB cell lines. All the cationic polymers showed a dosedependent effect on cytotoxicity. The slope of the dose-dependent cytotoxicity curve for PEI-25KD with KB cells was much steeper than those for PEI-CD·PEG-AD·FA-AD, Method A polyplexes, and Method B polyplexes, indicating much lower cytotoxicity of PEI-CD·PEG-AD·FA-AD, Method A polyplexes, and Method B polyplexes. However, there was a slight difference between PEI-CD and PEI-CD·PEG-AD·FA-AD in terms of dosedependent cytotoxicity. The low cytotoxicity of PEI-CD·PEGAD·FA-AD might be attributable to the introduction of PEG-AD and FA-AD, both of which result in a lower relative amino den-
Figure 7. Cell viability in the presence of PEI-CD (– ·^· –), PEI-CD·PEG-AD·FAAD (a a~a), Method A polyplexes (···*···) and Method B polyplexes (—&—) relative to that in the presence of PEI-25KD (—~—) in KB cell lines, as determined by MTT assay. The KB cells were treated with polymers of various nitrogen concentrations for 48 h in a serum-containing medium.
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The in vitro transfection efficiencies of the polyplexes formed by the two methods were assessed with the aid of luciferase as a marker gene in 10 % serum-supplemented medium or serum-free medium. The luciferase expression in the two polyplexes, relative to PEI-CD, naked pDNA (ND), and commercially available PEI-25KD, was quantified by means of a luciferase activity assay. Gene transfection mediated by cationic polymers was carried out in FR-negative HEK293 and FR-positive KB cells as described in the Experimental. In FR-positive KB cells (Figure 8) the gene transfection efficiency followed the order
Figure 8. In vitro gene transfection efficiencies of the polycation·pRL-CMV polyplexes at an N/P ratio of 20 both in 10 % serum-supplemented medium and in serum-free medium with HEK293 and KB cell lines. Naked DNA(ND) and PEI-25KD were used as controls.
Method A > Method B > PEI-CD. The gene transfection efficiency of Method A polyplexes was almost equal to that of PEI25KD. The transfection efficiency of the folic-acid-modified polymers was increased dramatically in FR-positive KB cells, probably due to FR-mediated cellular uptake. The transfection efficiency of Method A polyplexes was also higher than that of Method B polyplexes. The only difference between Method A and Method B was the self-assembly schemes (Figure 1). A possible reason was that the average particle size of Method A polyplexes was less than that of Method B polyplexes, and the surfaces of Method A polyplexes thus had proportionately more folic acid. Additionally, the transfection efficiency of PEICD was lower than those of the Method A and Method B polyplexes. A possible cause of the low-level transfection of PEI-CD was that poorly internalized large aggregates were formed because of the absence of sterically stabilizing PEG segments on the surfaces of the polyplexes.[10] Therefore, polyplexes produced by Method A could efficiently deliver pDNA into FR-positive cells through FR-mediated cellular uptake. However, in FR-negative HEK293 cells, FR-mediated cellular uptake was impossible, and the FA showed no positive effect
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Full Papers on the gene transfection efficiency. Similarly, the gene transfection efficiency followed the order Method A > Method B > PEICD. A possible reason was that the smaller sizes of the polyplexes might have resulted in an increase in the extent of cellular internalization of the particles, thereby leading to better transfection efficiencies for polyplexes produced by Method A. As shown in Figure 8, we also observed that the transfection efficiency of PEI-CD displayed changes in different environments (10 % serum-supplemented or serum-free medium). However, the transfection efficiencies of the polyplexes produced by Method A and by Method B showed little effect. The reason was that PEG-AD had been introduced onto both sets of polyplexes, because PEGylation can effectively improve serum stability. To demonstrate the targeting effect of FA-grafted carriers further, competition tests were carried out by addition of free FA at different concentrations (0.001, 0.010, and 0.100 mol L¢1) to the cell culture medium during the gene transfection. As shown in Figure 9 A, the gene transfection efficiencies of polyplexes produced by both Methods A and B decreased with increasing free FA concentration in FR-positive KB cells. In contrast, as shown in Figure 9 B, there were no significant changes in gene transfection efficiency for either carrier, produced by either Method A or B, in FR-negative HEK293 cells. This outstanding results seen for the polyplexes produced by Method A as gene carrier are that: they are assembled from FA-AD, which can be recognized by target cells more readily than PEI-CD, they have a better complexation capability than the polyplexes produced by Method B, they are taken up into the cells easily, due to their nanoparticle character, and they
Figure 9. In vitro gene transfection efficiencies of the polycation·pRL-CMV polyplexes at an N/P ratio of 20 in A) KB, and B) HEK293 cells in RPMI 1640 medium treated with FA at different concentrations (0.000, 0.001, 0.010, and 0.100 mol L¢1). Naked DNA(ND) and PEI-25KD were used as controls.
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have no significant cytotoxicity, relative to PEI-25KD, in the target cells.
Conclusions In this study, a gene-delivery system offering low cytotoxicity and efficient targeting has been developed, based on the selfassembly of PEI-CD with FA-AD and PEG-AD. Polyplexes produced by Method A are capable of condensing pDNA into nanoparticles in the < 200 nm size range by means of a “multilayer bricks-mortar” strategy. Importantly, the transfection efficiency of the polyplexes produced by Method A is comparable to that of PEI-25KD but with lower cytotoxicity in KB cells. The “multilayer bricks-mortar” strategy for self-assembly gene carriers is worthy of further investigation.
Experimental Section Materials: b-Cyclodextrin (b-CD), potassium carbonate (K2CO3), branched polyethylenimine (PEI) with average molecular weights of 600 Da and 25 kDa, poly(ethylene glycol) (PEG, 2 kDa), 4-dimethylaminopyridine (DMAP), N,N’-dicyclohexylcarbodiimide (DCC), adamantylamine, folic acid (FA), N-hydroxysuccinimide (NHS), and 1-(3dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) were purchased from Aladdin Industrial Corporation (Shanghai, China). All solvents were reagent grade, purchased from commercial sources. All experiments were carried out with ultrapure water. Synthesis of PEI-CD: The PEI-CD conjugate was synthesized according to the previous literature.[16] Briefly, mono-6-O-(p-tolylsulfonyl)-b-CD (b-CD-OTs, 2.6 g, 2.0 mmol), branched PEI 600D (0.06 g, 0.1 mmol), and potassium carbonate (0.5 g, 4 mmol) were dissolved in DMSO (40 mL). The mixture was stirred at 85 8C for 24 h and then precipitated in acetone. Subsequently, the precipitate was dissolved in water and transferred to a dialysis bag (MWCO, 2 kDa) and dialyzed against deionized water for 96 h. After dialysis, the resultant solution was concentrated under reduced pressure to a few milliliters before pouring into acetone (100 mL). A fine yellow precipitate was formed and gathered by filtration. The pure PEI-CD was further dried in vacuum at 40 8C overnight. Yield: 62 %. 1 H NMR (D2O, 400 MHz): dH = 4.75 (H1 of CD), 3.40–3.78 (H3, H5, H6 of CD), 3.00–3.40 (H2, H4 of CD), 2.12–2.76 ppm (-CH2- of PEI). Synthesis of PEG-AD: A solid-state mixture of PEG-2K (2.0 g, 1.0 mmol) and DMAP (0.5 g, 4.0 mmol) was dried under vacuum at 40 8C overnight. After the mixture had been cooled to room temperature, a solution of adamantane carboxylic acid (0.4 g, 2.0 mmol) in anhydrous CH2Cl2 (10 mL) was added with stirring. Subsequently, a solution of DCC (5 mL, 1 mol L¢1) in CH2Cl2 was added to the reaction mixture dropwise with stirring over a period of 5 min. Several minutes later, the by-product dicyclohexylurea (DCU) began to precipitate as a white powder. After being stirred for another 48 h at room temperature, the final reaction mixture was filtered to remove insoluble DCU. The filtrate was concentrated to ca. 5 mL and precipitated with ether (500 mL) to afford the product as a white powder. Yield: 75 %. 1H NMR (D2O, 400 MHz): dH = 3.44 (-CH2- of PEG), 0.85–1.75 ppm (protons of AD); 13C NMR (D2O, 400 MHz): d = 168.3, 72.8, 71.7, 70.6, 61.9, 59.4, 41.7, 36.1, 29.3 ppm; MS (MALDI-TOF): calcd: 2163.24 [M+ +H] + ; found: 2162.97. Synthesis of FA-AD: NHS (0.3 g, 2.4 mmol) and EDC (0.5 g, 2.4 mmol) were added to a solution of folic acid (0.9 g, 2 mmol) in
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Full Papers DMF (25 mL). The resulting mixture was stirred in the ice bath for 30 min to give a yellow precipitate. Subsequently, a solution of 1adamantylamine (0.3 g, 2.1 mmol) in DMF (5.0 mL) was added, and the resulting mixture was allowed to warm to room temperature and then to react for another 24 h. The reaction solution was poured into acetone/water (1:4, v/v, 300 mL) to form a precipitate. The precipitate was filtered, washed with acetone, and dried under vacuum at 40 8C for 24 h. Yield: 83 %. 1H NMR ([D6]DMSO, 400 MHz): dH = 8.63 (1 H), 7.92 (1 H; -PhCONH-), 7.61 (2 H; protons on Ph close to carbonyl group), 7.22 (1 H ;-CONH-AD), 6.89 (3 H; ¢ NH2 and -CH2NHPh-), 6.60 (2 H; protons on Ph close to secondary amine), 4.48 (2 H; -CH2NHPh-), 4.12–4.37 (m, 1 H; -CONHCHCOOH), 2.08–2.35 (2 H; -CHCH2CH2CONH-AD), 1.95 (2 H; -CHCH2CH2CONHAD), 1.35–1.90 ppm (15 H; protons of AD); 13C NMR ([D6]DMSO, 400 MHz): d = 175.2, 174.9, 171.3, 166.7, 162.6, 157.1, 154.6, 150.9, +H] + ; 149.2, 129.5, 127.9, 122.0, 111.3 ppm. HRMS: calcd: 575.27 [M+ found: 575.25. Determination of degree of substitution: The number of grafted CD units per PEI (600D) unit, here defined as the degree of substitution (DS), was calculated by 1H NMR integration. The DS was determined by comparison of the integrals of the H1 protons of b-CD and of the methylene protons of PEI as previously described. The mean DS value for PEI-CD was 6. Plasmid: The plasmid DNA was amplified in Escherichia coli and purified by use of a commercial kit according to the supplier’s protocol (Qiagen). The purity and concentration of the purified plasmid DNA were determined by electrophoresis in agarose gel (1 %) and by absorption at 260 and 280 nm. The purified plasmid DNA was resuspended in TE buffer [Tris·Cl (10 mm, pH 7.5), EDTA (1 mm)] and kept in aliquots at 150 ng mL¢1. Stoichiometry: n ¼ N=P,
C¼
n P M X
where N was the concentration of nitrogen in cationic compounds [nmol mL¢1], P was the concentration of phosphorus in pDNA [nmol mL¢1], X was the number of nitrogen atoms in cationic compounds, M was the molecular weight of cationic compounds [g mol¢1], and C was concentration of cationic compounds [ng mL¢1]. The molecular weight of a typical DNA base is 325 g mol¢1. One molecule of DNA contains one phosphorus atom, so 1 mg of DNA corresponds to 3 nmol of phosphorus. Preparation of the polyplexes by Method A: Plasmid DNA (pDNA) stock solution was diluted to a chosen concentration (50 ng mL¢1, P = 0.15 nmol mL¢1). PEI-CD (1.0 equiv, 1564.29 ng mL¢1, 1 equiv PEI-CD contains 6 equiv CD) was added dropwise to the pDNA solution in equivalent volume according to the predetermined nitrogen/phosphorus (N/P = 20) ratios. The mixture solution was vortexed and incubated for 30 min at room temperature. Subsequently, the PEG-AD (2.0 equiv, 926.15 ng mL¢1) and FA-AD (2.0 equiv, 246.32 ng mL¢1) solutions were slowly injected into the mixture, followed by stirring of the solution mixture for 30 min at room temperature and for the subsequent experiments. The amount of CD was more than the amount of PEG-AD and FA-AD. The purpose was to avoid free FA-AD. Preparation of the polyplexes by Method B: PEG-AD (2.0 equiv, 926.15 ng mL¢1) and FA-AD (2.0 equiv, 246.32 ng mL¢1) were mixed, and then the PEI-CD (1.0 equiv, 1564.29 ng mL¢1, 1 equiv PEI-CD contains 6 equiv CD) was slowly injected into the mixture, followed by stirring of the solution mixture for 30 min at room temperature. ChemBioChem 2015, 16, 1622 – 1628
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Subsequently, the solution mixture was added dropwise to pDNA solution (0.15 nmol mL¢1) in equivalent volume according to the predetermined nitrogen/phosphorus (N/P = 20) ratios. The mixture solution was vortexed and incubated for 30 min at room temperature and for the subsequent experiments. The amount of CD was more than the amount of PEG-AD and FA-AD. The purpose was to avoid free FA-AD. Biophysical properties of the polyplexes prepared by both methods: Polycation to pDNA ratios are expressed as molar ratios of nitrogen in polycation to phosphate (P) in pDNA (or as N/P ratios). The ability of each polycation to bind pDNA at various N/P ratios was examined through agarose gel electrophoresis. In general, the polyplexes were prepared by adding polymer solution (10 mL) dropwise to an equal volume of plasmid solution containing pDNA (0.5 mg), followed by stirring of the solution mixture for 10 min and incubation for 30 min at room temperature. After mixing, the polyplex solution was analyzed on agarose gel (0.9 %) containing ethidium bromide (0.5 mg mL¢1). Gel electrophoresis was carried out in 1 Õ Tris acetate/EDTA (TAE) buffer (Tris acetate (40 mm), EDTA (1 mm)) at 100 V for 30 min in a Sub-Cell system. pDNA bands were visualized with a UV lamp and use of a Gel Doc system. The sizes and size distributions of the Method A and B polyplexes were evaluated by dynamic light scattering with a particle size analyzer at room temperature with a scattering angle of 908. AFM imaging of the nanoparticles was conducted in tapping mode with dry samples on mica. The AFM tips had a typical radius of 7 nm or less, and the images were recorded with a scan rate of 0.5 or 1 Hz. Samples were prepared by dropping the solution (2 mL) onto a mica surface, followed by 30 min drying at 20 8C. In vitro cytotoxicity studies: Cells were suspended in RPMI 1640 supplemented with fetal bovine serum (Hyclone, 10 %) and seeded into the wells of 96-well culture plates at a density of 1 Õ 104/ 100 mL medium. After 24 h preincubation under a humidified CO2 (5 %) atmosphere at 37 8C, new media (the culture medium described above, containing different concentrations of polycation) were added, and culture was continued for a further 48 h. Cell viability was evaluated by MTT assay. Briefly, MTT stock solution (5 mg mL¢1 in PBS, 20 mL) was added to cell cultures (100 mL) in 96well culture plates for 4 h incubation at 37 8C. Plates were then centrifuged, and MTT-containing medium culture was removed. Precipitated formazan was dissolved in DMSO (150 mL). Results were read within 15 min in a spectrometer at 490 nm, and the means of triplicates were calculated. In vitro transfection and luciferase assay: Transfection studies were performed in HEK293 and KB cells with use of the plasmid pRL-CMV as reporter gene. Cells were cultured in complete Dulbecco’s modified Eagle’s medium (DMEM) supplemented with FBS (10 %) at 37 8C, under CO2 (5 %), and at 95 % relative humidity. Cells were seeded in 24-well plates at a density of 5 Õ 104 cells per well. After 48 h, the culture medium was replaced with medium (10 % serum-supplemented or serum-free) containing the transfection polyplexes prepared at an N/P ratio of 20. The cells were incubated with the transfection polyplexes for 12 h, after which the medium was replaced with fresh medium (500 mL) supplemented with FBS (10 %), and the cells were further incubated for an additional 12 h under the same conditions, resulting in a total transfection time of 24 h. Cells were washed with PBS, trypsinized, and analyzed. Luciferase gene expression was quantified by using a commercial kit and a luminometer. Protein concentrations in the samples were analyzed by use of a bicinchoninic acid assay. Absorption was measured with a microplate reader at 570 nm and compared to a standard curve calibrated with BSA samples of known concentration.
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Full Papers Results are expressed as relative light units per milligram of cell protein lysate (RLU/mg protein).
Acknowledgements This work was supported by the National Natural Science Foundation of China (NNSFC) (No. 21362016) and the Natural Science Foundation of Yunnan Province (No. 2011FZ059), which are gratefully acknowledged. Keywords: cyclodextrins · drug delivery · gene carriers · polyethylenimine · self-assembly [1] a) M. C. Filion, N. C. Phillips, Int. J. Pharm. 1998, 162, 159 – 170; b) Y. Kodama, T. Nakamura, T. Kurosaki, K. Egashira, T. Mine, H. Nakagawa, T. Muro, T. Kitahara, N. Higuchi, H. Sasaki, Eur. J. Pharm. Biopharm. 2014, 87, 472 – 479. [2] Y. Ping, Q. D. Hu, G. P. Tang, J. Li, Biomaterials 2013, 34, 6482 – 6494. [3] D. W. Pack, A. S. Hoffman, S. Pun, P. S. Stayton, Nat. Rev. Drug Discovery 2005, 4, 581 – 593. [4] O. Boussif, F. Lezoualc’h, M. A. Zanta, M. D. Mergny, D. Scherman, B. Demeneix, J. P. Behr, Proc. Natl. Acad. Sci. USA 1995, 92, 7297 – 7301. [5] M. Neu, D. Fischer, T. Kissel, J. Gene Med. 2005, 7, 992 – 1009. [6] M. Ogris, S. Brunner, S. Schuller, R. Kircheis, E. Wagner, Gene Ther. 1999, 6, 595 – 605.
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[7] a) H. Yao, S. S. Ng, W. O. Tucker, Y. K. T. Tsang, K. Man, X. M. Wang, B. K. C. Chow, H. F. Kung, G. P. Tang, M. C. Lin, Biomaterials 2009, 30, 5793 – 5803; b) G. Zuber, L. Zammut-Italiano, E. Dauty, J. P. Behr, Angew. Chem. Int. Ed. 2003, 42, 2666 – 2669; Angew. Chem. 2003, 115, 2770 – 2773. [8] P. Garin-Chesa, I. Campbell, P. E. Saigo, J. L. Lewis, Jr., L. J. Old, W. J. Rettig, Am. J. Pathol. 1993, 142, 557 – 567. [9] a) J. W. Zhou, H. Ritter, Polym. Chem. 2010, 1, 1552 – 1559; b) R. Liao, M. Liu, X. Liao, B. Yang, Prog. Chem. 2015, 27, 79 – 90; c) R. Liao, Y. Zhao, X. Liao, M. Liu, C. Gao, J. Yang, B. Yang, Polym. Adv. Technol. 2015, 26, 487 – 494. [10] A. Kulkarni, W. Deng, S. H. Hyun, D. H. Thompson, Bioconjugate Chem. 2012, 23, 933 – 940. [11] M. E. Davis, J. E. Zuckerman, C. H. J. Choi, D. Seligson, A. Tolcher, C. A. Alabi, Y. Yen, J. D. Heidel, A. Ribas, Nature 2010, 464, 1067 – U1140. [12] Y. Yang, Y. M. Zhang, Y. Chen, J. T. Chen, Y. Liu, J. Med. Chem. 2013, 56, 9725 – 9736. [13] S. H. Pun, M. E. Davis, Bioconjugate Chem. 2002, 13, 630 – 639. [14] M. Ogris, P. Steinlein, S. Carotta, S. Brunner, E. Wagner, AAPS Pharm. Sci. 2001, 3, E21. [15] S. Mishra, P. Webster, M. E. Davis, Eur. J. Cell Biol. 2004, 83, 97 – 111. [16] R. J. Dong, H. Y. Chen, D. L. Wang, Y. Y. Zhuang, L. J. Zhu, Y. Su, D. Y. Yan, X. Y. Zhu, ACS Macro Lett. 2012, 1, 1208 – 1211.
Manuscript received: April 22, 2015 Accepted article published: May 28, 2015 Final article published: June 11, 2015
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Folic-Acid-Targeted Self-Assembling Supramolecular Carrier for Gene Delivery Rongqiang Liao,[a] Shouhui Yi,[b] Manshuo Liu,[a] Wenling Jin,[a] and Bo Yang*[a] A targeting gene carrier for cancer-specific delivery was successfully developed through a “multilayer bricks-mortar” strategy. The gene carrier was composed of adamantane-functionalized folic acid (FA-AD), an adamantane-functionalized poly(ethylene glycol) derivative (PEG-AD), and b-cyclodextrin-grafted low-molecular-weight branched polyethylenimine (PEI-CD). Carriers produced by two different self-assembly schemes, involving either precomplexation of the PEI-CD with the FA-AD and PEG-AD before pDNA condensation (Method A) or pDNA condensation with the PEI-CD prior to addition of the FA-AD
and PEG-AD to engage host–guest complexation (Method B) were investigated for their ability to compact pDNA into nanoparticles. Cell viability studies show that the material produced by the Method A assembly scheme has lower cytotoxicity than branched PEI 25 kDa (PEI-25KD) and that the transfection efficiency is maintained. These findings suggest that the gene carrier, based on multivalent host–guest interactions, could be an effective, targeted, and low-toxicity carrier for delivering nucleic acid to target cells.
Introduction In gene carriers, cationic polymers possess several advantages: they have, for example, high transfection capability and low toxicity, they are non-immunogenic, and their chemical and structural properties can be tightly controlled. This allows carriers that are suitable for mass production to be designed.[1] Cationic polymers can spontaneously interact with nucleic acids to form polyplexes through electrostatic interactions. These polyplexes are usually 50–200 nm nanoparticles with positive surface charge; they are thought to interact electrostatically with negatively charged cell membranes to promote cellular uptake through nonspecific endocytosis.[2] Of those cationic polymers, polyethylenimine (PEI) is a promising delivery reagent, primarily because of its excellent transfection efficiency in a wide range of cell types.[3] By virtue of its abundant primary amine groups, PEI readily forms polyplexes with negatively charged nucleic acids and subsequently buffers the endosomal environment, facilitating the release of nucleic acid in the cytosol.[4] However, it should be noted that the transfection efficiency and cytotoxicity of PEI are dependent on its molecular weight. In general, PEI with higher molecular weight has a higher transfection efficiency, but also higher cytotoxicity. The primary reason for the cytotoxicity is that the positive charge on a polyplex can also interact with a wide variety of negatively charged components in vivo.[5] Consequent[a] R. Liao, M. Liu, W. Jin, Prof. B. Yang Faculty of Life Science and Technology Kunming University of Science and Technology Kunming 650500 (P. R. China) E-mail: [email protected] [email protected] [b] S. Yi Department of Oncology, Chongqing Cancer Hospital Chongqing 400011 (P. R. China)
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ly, shielding agents have to be introduced onto polyplex particle surfaces in order to prevent such nonspecific interactions. One common approach to shielding excessive positive charge is to attach poly(ethylene glycol) (PEG) chains onto the polyplex particle surface. This strategy, often termed PEGylation, can effectively improve serum stability and reduce cytotoxicity.[6] In addition to modification of cationic polymers, gene carriers—a variety of different target-specific ligands, including various transfer proteins and small-molecule compounds such as folic acid (FA)—have also been explored with the goal of enhancing target-specific gene delivery.[7] In recent years, FA has been widely utilized as a promising targeting ligand because folate receptors (FRs) are overexpressed in many human tumor cells, including malignancies of the colon, rectum, ovary, kidney, breast, and lung, but have little expression in normal tissues.[8] Similarly, b-cyclodextrin (CD) is widely used for gene delivery because of its derivatives’ excellent properties such as low toxicity, high biodegradability, stability, and biocompatibility and facile chemical modification.[9] Additionally, CD has been used as a convenient building block for the construction of new functional and nanostructured materials. A variety of CD-based systems such as CD polymers, CD polyrotaxanes, and CD dendrimers are promising materials for nonviral vector development.[10] Recently, Davis reported a diverse class of CD oligomers coupled through cationic linkers, and successfully used them as vectors for siRNA delivery in a clinical trial for treatment of melanoma in humans.[11] CD can host various poorly soluble molecules in its hydrophobic cavity, with high size- and shape-selectivity both in aqueous solution and in the solid state.[12] With some molecules, this association can be quite strong. For example, the association constants (Ka) for adaman-
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Full Papers tane (AD) derivatives and CD are of the order of 104– 105.[13] This noncovalent self-assembly method has many advantages, such as the lack of need for special reaction equipment, mild reaction conditions, simplicity of operation, environmental friendliness, and easy batch preparation. The AD guest molecule can be PEGylated with PEG to improve serum stability and to reduce cytotoxicity, and can be vectorized with FA to target folate receptors on the tumor cell surface. Introduction of a gene vector based on CD onto such adamantane-functionalized molecules can improve the transfection efficiency significantly. Here we have employed this supramolecular strategy to achieve self-assembly of a nucleic acid delivery vector from three different molecular building blocks: namely, 1) b-cyclodextrin-grafted low-molecular-weight branched polyethylenimine (PEI-CD), 2) adamantane-functionalized folate (FA-AD), and 3) an adamantane-functionalized poly(ethylene gly- Figure 2. 1H NMR spectrum of PEI-CD. col) derivative (PEG-AD). It was anticipated that plasmid DNA (pDNA) compaction might be achievable through multivalent electrostatic interactions between the PEIof the gene delivery systems. PEI-CD was synthesized by nucleCD and the pDNA, and that the CD units in the PEI-CD could ophilic substitution of the tosyloxy group in b-CD-OTs by an host the AD groups of the FA-AD and PEG-AD through hydroamino group of PEI in the presence of potassium carbonate as phobic interactions. This design could enable the compaction the catalyst. As shown in Figure 2, the CD protons displayed of pDNA into stable nanometer-sized particles that can then chemical shifts at d = 4.7 ppm and 3.0–3.8 ppm, distinct from be targeted and internalized within tumor cells through folate those of the PEI protons (d = 2.0–2.7 ppm). This result indicated receptor recognition at the tumor cell surface (Figure 1). that the PEI had been linked to the CD. In the PEI-CD molecule there were about six CD units grafted on a branched PEI backbone according to the 1H NMR spectrum. The successful preparation of PEI-CD·PEG-AD·FA-AD complexes was also clearly demonstrated by the 1H NMR spectrum shown in Figure 3. AD was insoluble in H2O, but there were AD proton peaks in the system in D2O. This result indicated that the AD was bound in the CD cavity. To further demonstrate the presence of the AD in the CD cavity, the chemical shifts (d) of the complexes were compared (Table 1). After inclusion complexation with AD, the H3 proton of CD had shifted by 0.110 ppm, the H5 proton of CD by 0.090 ppm, and the protons of AD by ca. 0.400 ppm. These results thus provided further evidence that the AD was included in the CD cavity.
Table 1. The chemical shifts (d) of AD, PEI-CD, and the AD·PEI-CD complex.
Figure 1. Conceptual diagram of polycation/pDNA complexation.
Results and Discussion Preparation of PEI-CD, PEG-AD, FA-AD, and complexes In this work, we constructed the folate-targeted gene vector. The PEI-CD·PEG-AD·FA-AD complexes to be used as the final gene carriers in this study could be simply obtained by a “bricks and mortar” strategy. The self-assembly process should facilitate future quality control and manufacturing procedures ChemBioChem 2015, 16, 1622 – 1628
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H1 of CD H2 of CD H3 of CD H4 of CD H5 of CD H6 of CD H1 of AD H2 of AD H3 of AD
d dd dd dd m dd m d m
AD
PEI-CD
Complex
– – – – – – 1.645 1.805 1.925
4.710 3.275 3.590 3.225 3.515 3.545 – – –
4.725 3.305 3.700 3.335 3.605 3.595 1.125 1.455 1.565
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Full Papers Zeta potential is an indicator of surface charge on a polycation/pDNA nanoparticle, and a positive surface charge on a nanoparticle could electrostatically interact with a negatively charged cellular membrane. As indicated in Figure 5 B, the zeta potentials of Method A polyplexes have very low values, in the 0.5–2.5 mV range, regardless of N/P ratio, whereas the surface charges of Method B polyplexes increased from 0.8 to 6.9 mV with increasing N/P ratio. This might be caused by the neutral FA-AD and PEG-AD shielding the surface charge of PEI-CD·pDNA polyplexes for Method A. Figure 6 shows representative AFM images of the Method A and Method B polyplexes at an N/P ratio of 20. The AFM images distinctly revealed that most of the tight polyplexes produced by either of the two methods had average diameters of 200 nm or
Figure 3. 1H NMR spectrum of PEI-CD·PEG-AD·FA-ADa.
Biophysical characterization of the polyplexes obtained by both methods A successful gene delivery system requires that the nucleic acid be compacted by the polycation into nanoparticles small enough to facilitate cellular uptake.[14] In this work, the ability of PEI-CD·PEG-AD·FA-AD to condense pDNA into particulate structures was confirmed by agarose gel electrophoresis, measurement of zeta potentials, and atomic force microscopy (AFM). Two different complexation methods were used to evaluate the relative capacities of PEI-CD·PEG-AD·FA-AD host– guest polymer assemblies for pDNA condensation. In Method A, the pDNA was first complexed with the PEI-CD, followed by addition of the PEG-AD and FA-AD. In Method B, PEG-AD and FA-AD were preassociated with PEI-CD before addition to the pDNA solution. Gel-shift assays of the pDNA polyplexes with PEI-CD·PEGAD·FA-AD assemblies indicated that the two methods of formulation gave rise to slightly different pDNA complexation abilities. As shown in Figure 4, the polyplexes formed by Method A were able to inhibit DNA migration at an N/P ratio of 3. In the case of the polyplexes formed by Method B, slightly reduced pDNA complexation capability was observed, with an N/P ratio of 10 required to inhibit pDNA mobility completely. Due to partial shielding of surface positive charge and steric hindrance by the premixed FA-AD and PEG-AD towards PEICD, larger quantities of Method B PEI-CD·PEG-AD·FA-AD were generally necessary to achieve pDNA complexation than in Method A. Condensation of pDNA into nanoparticles in the size range of 50–200 nm was critical not only for in vivo distribution, but also for efficient cellular uptake by endocytosis through clathrin-coated pit mechanisms in cultured cells.[15] The effect of N/P ratio on particle size of complexes formed by both methods was investigated. As shown in Figure 5 A, the particle sizes of the two complexes decreased with increasing N/P ratio. Both methods could form nanoparticles of < 200 nm in average diameter at an N/P ratio of 30. However, the particle sizes of the Method B polyplexes were slightly larger than those of the Method A polyplexes. ChemBioChem 2015, 16, 1622 – 1628
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Figure 4. Agarose gel electrophoresis retardation of pDNA migration by polyplexes produced by Method A or Method B at various N/P ratios.
Figure 5. A) Particle size and B) zeta potential of polyplexes formed by both Methods A (~) and B (&).
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Full Papers sity. Meanwhile, the cytotoxicities of Method A polyplexes and Method B polyplexes showed no great difference. The main reason was also the introduction of PEG-AD and FA-AD.
Transfection efficiencies of the polyplexes produced by the two methods Figure 6. AFM images of A) pDNA, and of polyplexes produced by B) Method A, and C) Method B (N/P ratio of 20).
less; this was consistent with the results of the particle size measurement. In vitro cytotoxicity Gene carrier cytotoxicity has a great influence on transfection efficiency for pDNA delivery. In vitro toxicity study is essential to confirm the safety of any cationic polymers used for gene delivery. The primary reason for cytotoxicity is that the positive charge of a polyplex can also interact with a wide variety of negatively charged components in vivo. The presence of primary, secondary, and tertiary amines, which are usually charged under physiological conditions, is believed to induce gene vector cytotoxicity. Figure 7 shows cell viability as a function of cationic polymer concentration, determined by MTT assay in KB cell lines. All the cationic polymers showed a dosedependent effect on cytotoxicity. The slope of the dose-dependent cytotoxicity curve for PEI-25KD with KB cells was much steeper than those for PEI-CD·PEG-AD·FA-AD, Method A polyplexes, and Method B polyplexes, indicating much lower cytotoxicity of PEI-CD·PEG-AD·FA-AD, Method A polyplexes, and Method B polyplexes. However, there was a slight difference between PEI-CD and PEI-CD·PEG-AD·FA-AD in terms of dosedependent cytotoxicity. The low cytotoxicity of PEI-CD·PEGAD·FA-AD might be attributable to the introduction of PEG-AD and FA-AD, both of which result in a lower relative amino den-
Figure 7. Cell viability in the presence of PEI-CD (– ·^· –), PEI-CD·PEG-AD·FAAD (a a~a), Method A polyplexes (···*···) and Method B polyplexes (—&—) relative to that in the presence of PEI-25KD (—~—) in KB cell lines, as determined by MTT assay. The KB cells were treated with polymers of various nitrogen concentrations for 48 h in a serum-containing medium.
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The in vitro transfection efficiencies of the polyplexes formed by the two methods were assessed with the aid of luciferase as a marker gene in 10 % serum-supplemented medium or serum-free medium. The luciferase expression in the two polyplexes, relative to PEI-CD, naked pDNA (ND), and commercially available PEI-25KD, was quantified by means of a luciferase activity assay. Gene transfection mediated by cationic polymers was carried out in FR-negative HEK293 and FR-positive KB cells as described in the Experimental. In FR-positive KB cells (Figure 8) the gene transfection efficiency followed the order
Figure 8. In vitro gene transfection efficiencies of the polycation·pRL-CMV polyplexes at an N/P ratio of 20 both in 10 % serum-supplemented medium and in serum-free medium with HEK293 and KB cell lines. Naked DNA(ND) and PEI-25KD were used as controls.
Method A > Method B > PEI-CD. The gene transfection efficiency of Method A polyplexes was almost equal to that of PEI25KD. The transfection efficiency of the folic-acid-modified polymers was increased dramatically in FR-positive KB cells, probably due to FR-mediated cellular uptake. The transfection efficiency of Method A polyplexes was also higher than that of Method B polyplexes. The only difference between Method A and Method B was the self-assembly schemes (Figure 1). A possible reason was that the average particle size of Method A polyplexes was less than that of Method B polyplexes, and the surfaces of Method A polyplexes thus had proportionately more folic acid. Additionally, the transfection efficiency of PEICD was lower than those of the Method A and Method B polyplexes. A possible cause of the low-level transfection of PEI-CD was that poorly internalized large aggregates were formed because of the absence of sterically stabilizing PEG segments on the surfaces of the polyplexes.[10] Therefore, polyplexes produced by Method A could efficiently deliver pDNA into FR-positive cells through FR-mediated cellular uptake. However, in FR-negative HEK293 cells, FR-mediated cellular uptake was impossible, and the FA showed no positive effect
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Full Papers on the gene transfection efficiency. Similarly, the gene transfection efficiency followed the order Method A > Method B > PEICD. A possible reason was that the smaller sizes of the polyplexes might have resulted in an increase in the extent of cellular internalization of the particles, thereby leading to better transfection efficiencies for polyplexes produced by Method A. As shown in Figure 8, we also observed that the transfection efficiency of PEI-CD displayed changes in different environments (10 % serum-supplemented or serum-free medium). However, the transfection efficiencies of the polyplexes produced by Method A and by Method B showed little effect. The reason was that PEG-AD had been introduced onto both sets of polyplexes, because PEGylation can effectively improve serum stability. To demonstrate the targeting effect of FA-grafted carriers further, competition tests were carried out by addition of free FA at different concentrations (0.001, 0.010, and 0.100 mol L¢1) to the cell culture medium during the gene transfection. As shown in Figure 9 A, the gene transfection efficiencies of polyplexes produced by both Methods A and B decreased with increasing free FA concentration in FR-positive KB cells. In contrast, as shown in Figure 9 B, there were no significant changes in gene transfection efficiency for either carrier, produced by either Method A or B, in FR-negative HEK293 cells. This outstanding results seen for the polyplexes produced by Method A as gene carrier are that: they are assembled from FA-AD, which can be recognized by target cells more readily than PEI-CD, they have a better complexation capability than the polyplexes produced by Method B, they are taken up into the cells easily, due to their nanoparticle character, and they
Figure 9. In vitro gene transfection efficiencies of the polycation·pRL-CMV polyplexes at an N/P ratio of 20 in A) KB, and B) HEK293 cells in RPMI 1640 medium treated with FA at different concentrations (0.000, 0.001, 0.010, and 0.100 mol L¢1). Naked DNA(ND) and PEI-25KD were used as controls.
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have no significant cytotoxicity, relative to PEI-25KD, in the target cells.
Conclusions In this study, a gene-delivery system offering low cytotoxicity and efficient targeting has been developed, based on the selfassembly of PEI-CD with FA-AD and PEG-AD. Polyplexes produced by Method A are capable of condensing pDNA into nanoparticles in the < 200 nm size range by means of a “multilayer bricks-mortar” strategy. Importantly, the transfection efficiency of the polyplexes produced by Method A is comparable to that of PEI-25KD but with lower cytotoxicity in KB cells. The “multilayer bricks-mortar” strategy for self-assembly gene carriers is worthy of further investigation.
Experimental Section Materials: b-Cyclodextrin (b-CD), potassium carbonate (K2CO3), branched polyethylenimine (PEI) with average molecular weights of 600 Da and 25 kDa, poly(ethylene glycol) (PEG, 2 kDa), 4-dimethylaminopyridine (DMAP), N,N’-dicyclohexylcarbodiimide (DCC), adamantylamine, folic acid (FA), N-hydroxysuccinimide (NHS), and 1-(3dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) were purchased from Aladdin Industrial Corporation (Shanghai, China). All solvents were reagent grade, purchased from commercial sources. All experiments were carried out with ultrapure water. Synthesis of PEI-CD: The PEI-CD conjugate was synthesized according to the previous literature.[16] Briefly, mono-6-O-(p-tolylsulfonyl)-b-CD (b-CD-OTs, 2.6 g, 2.0 mmol), branched PEI 600D (0.06 g, 0.1 mmol), and potassium carbonate (0.5 g, 4 mmol) were dissolved in DMSO (40 mL). The mixture was stirred at 85 8C for 24 h and then precipitated in acetone. Subsequently, the precipitate was dissolved in water and transferred to a dialysis bag (MWCO, 2 kDa) and dialyzed against deionized water for 96 h. After dialysis, the resultant solution was concentrated under reduced pressure to a few milliliters before pouring into acetone (100 mL). A fine yellow precipitate was formed and gathered by filtration. The pure PEI-CD was further dried in vacuum at 40 8C overnight. Yield: 62 %. 1 H NMR (D2O, 400 MHz): dH = 4.75 (H1 of CD), 3.40–3.78 (H3, H5, H6 of CD), 3.00–3.40 (H2, H4 of CD), 2.12–2.76 ppm (-CH2- of PEI). Synthesis of PEG-AD: A solid-state mixture of PEG-2K (2.0 g, 1.0 mmol) and DMAP (0.5 g, 4.0 mmol) was dried under vacuum at 40 8C overnight. After the mixture had been cooled to room temperature, a solution of adamantane carboxylic acid (0.4 g, 2.0 mmol) in anhydrous CH2Cl2 (10 mL) was added with stirring. Subsequently, a solution of DCC (5 mL, 1 mol L¢1) in CH2Cl2 was added to the reaction mixture dropwise with stirring over a period of 5 min. Several minutes later, the by-product dicyclohexylurea (DCU) began to precipitate as a white powder. After being stirred for another 48 h at room temperature, the final reaction mixture was filtered to remove insoluble DCU. The filtrate was concentrated to ca. 5 mL and precipitated with ether (500 mL) to afford the product as a white powder. Yield: 75 %. 1H NMR (D2O, 400 MHz): dH = 3.44 (-CH2- of PEG), 0.85–1.75 ppm (protons of AD); 13C NMR (D2O, 400 MHz): d = 168.3, 72.8, 71.7, 70.6, 61.9, 59.4, 41.7, 36.1, 29.3 ppm; MS (MALDI-TOF): calcd: 2163.24 [M+ +H] + ; found: 2162.97. Synthesis of FA-AD: NHS (0.3 g, 2.4 mmol) and EDC (0.5 g, 2.4 mmol) were added to a solution of folic acid (0.9 g, 2 mmol) in
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Full Papers DMF (25 mL). The resulting mixture was stirred in the ice bath for 30 min to give a yellow precipitate. Subsequently, a solution of 1adamantylamine (0.3 g, 2.1 mmol) in DMF (5.0 mL) was added, and the resulting mixture was allowed to warm to room temperature and then to react for another 24 h. The reaction solution was poured into acetone/water (1:4, v/v, 300 mL) to form a precipitate. The precipitate was filtered, washed with acetone, and dried under vacuum at 40 8C for 24 h. Yield: 83 %. 1H NMR ([D6]DMSO, 400 MHz): dH = 8.63 (1 H), 7.92 (1 H; -PhCONH-), 7.61 (2 H; protons on Ph close to carbonyl group), 7.22 (1 H ;-CONH-AD), 6.89 (3 H; ¢ NH2 and -CH2NHPh-), 6.60 (2 H; protons on Ph close to secondary amine), 4.48 (2 H; -CH2NHPh-), 4.12–4.37 (m, 1 H; -CONHCHCOOH), 2.08–2.35 (2 H; -CHCH2CH2CONH-AD), 1.95 (2 H; -CHCH2CH2CONHAD), 1.35–1.90 ppm (15 H; protons of AD); 13C NMR ([D6]DMSO, 400 MHz): d = 175.2, 174.9, 171.3, 166.7, 162.6, 157.1, 154.6, 150.9, +H] + ; 149.2, 129.5, 127.9, 122.0, 111.3 ppm. HRMS: calcd: 575.27 [M+ found: 575.25. Determination of degree of substitution: The number of grafted CD units per PEI (600D) unit, here defined as the degree of substitution (DS), was calculated by 1H NMR integration. The DS was determined by comparison of the integrals of the H1 protons of b-CD and of the methylene protons of PEI as previously described. The mean DS value for PEI-CD was 6. Plasmid: The plasmid DNA was amplified in Escherichia coli and purified by use of a commercial kit according to the supplier’s protocol (Qiagen). The purity and concentration of the purified plasmid DNA were determined by electrophoresis in agarose gel (1 %) and by absorption at 260 and 280 nm. The purified plasmid DNA was resuspended in TE buffer [Tris·Cl (10 mm, pH 7.5), EDTA (1 mm)] and kept in aliquots at 150 ng mL¢1. Stoichiometry: n ¼ N=P,
C¼
n P M X
where N was the concentration of nitrogen in cationic compounds [nmol mL¢1], P was the concentration of phosphorus in pDNA [nmol mL¢1], X was the number of nitrogen atoms in cationic compounds, M was the molecular weight of cationic compounds [g mol¢1], and C was concentration of cationic compounds [ng mL¢1]. The molecular weight of a typical DNA base is 325 g mol¢1. One molecule of DNA contains one phosphorus atom, so 1 mg of DNA corresponds to 3 nmol of phosphorus. Preparation of the polyplexes by Method A: Plasmid DNA (pDNA) stock solution was diluted to a chosen concentration (50 ng mL¢1, P = 0.15 nmol mL¢1). PEI-CD (1.0 equiv, 1564.29 ng mL¢1, 1 equiv PEI-CD contains 6 equiv CD) was added dropwise to the pDNA solution in equivalent volume according to the predetermined nitrogen/phosphorus (N/P = 20) ratios. The mixture solution was vortexed and incubated for 30 min at room temperature. Subsequently, the PEG-AD (2.0 equiv, 926.15 ng mL¢1) and FA-AD (2.0 equiv, 246.32 ng mL¢1) solutions were slowly injected into the mixture, followed by stirring of the solution mixture for 30 min at room temperature and for the subsequent experiments. The amount of CD was more than the amount of PEG-AD and FA-AD. The purpose was to avoid free FA-AD. Preparation of the polyplexes by Method B: PEG-AD (2.0 equiv, 926.15 ng mL¢1) and FA-AD (2.0 equiv, 246.32 ng mL¢1) were mixed, and then the PEI-CD (1.0 equiv, 1564.29 ng mL¢1, 1 equiv PEI-CD contains 6 equiv CD) was slowly injected into the mixture, followed by stirring of the solution mixture for 30 min at room temperature. ChemBioChem 2015, 16, 1622 – 1628
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Subsequently, the solution mixture was added dropwise to pDNA solution (0.15 nmol mL¢1) in equivalent volume according to the predetermined nitrogen/phosphorus (N/P = 20) ratios. The mixture solution was vortexed and incubated for 30 min at room temperature and for the subsequent experiments. The amount of CD was more than the amount of PEG-AD and FA-AD. The purpose was to avoid free FA-AD. Biophysical properties of the polyplexes prepared by both methods: Polycation to pDNA ratios are expressed as molar ratios of nitrogen in polycation to phosphate (P) in pDNA (or as N/P ratios). The ability of each polycation to bind pDNA at various N/P ratios was examined through agarose gel electrophoresis. In general, the polyplexes were prepared by adding polymer solution (10 mL) dropwise to an equal volume of plasmid solution containing pDNA (0.5 mg), followed by stirring of the solution mixture for 10 min and incubation for 30 min at room temperature. After mixing, the polyplex solution was analyzed on agarose gel (0.9 %) containing ethidium bromide (0.5 mg mL¢1). Gel electrophoresis was carried out in 1 Õ Tris acetate/EDTA (TAE) buffer (Tris acetate (40 mm), EDTA (1 mm)) at 100 V for 30 min in a Sub-Cell system. pDNA bands were visualized with a UV lamp and use of a Gel Doc system. The sizes and size distributions of the Method A and B polyplexes were evaluated by dynamic light scattering with a particle size analyzer at room temperature with a scattering angle of 908. AFM imaging of the nanoparticles was conducted in tapping mode with dry samples on mica. The AFM tips had a typical radius of 7 nm or less, and the images were recorded with a scan rate of 0.5 or 1 Hz. Samples were prepared by dropping the solution (2 mL) onto a mica surface, followed by 30 min drying at 20 8C. In vitro cytotoxicity studies: Cells were suspended in RPMI 1640 supplemented with fetal bovine serum (Hyclone, 10 %) and seeded into the wells of 96-well culture plates at a density of 1 Õ 104/ 100 mL medium. After 24 h preincubation under a humidified CO2 (5 %) atmosphere at 37 8C, new media (the culture medium described above, containing different concentrations of polycation) were added, and culture was continued for a further 48 h. Cell viability was evaluated by MTT assay. Briefly, MTT stock solution (5 mg mL¢1 in PBS, 20 mL) was added to cell cultures (100 mL) in 96well culture plates for 4 h incubation at 37 8C. Plates were then centrifuged, and MTT-containing medium culture was removed. Precipitated formazan was dissolved in DMSO (150 mL). Results were read within 15 min in a spectrometer at 490 nm, and the means of triplicates were calculated. In vitro transfection and luciferase assay: Transfection studies were performed in HEK293 and KB cells with use of the plasmid pRL-CMV as reporter gene. Cells were cultured in complete Dulbecco’s modified Eagle’s medium (DMEM) supplemented with FBS (10 %) at 37 8C, under CO2 (5 %), and at 95 % relative humidity. Cells were seeded in 24-well plates at a density of 5 Õ 104 cells per well. After 48 h, the culture medium was replaced with medium (10 % serum-supplemented or serum-free) containing the transfection polyplexes prepared at an N/P ratio of 20. The cells were incubated with the transfection polyplexes for 12 h, after which the medium was replaced with fresh medium (500 mL) supplemented with FBS (10 %), and the cells were further incubated for an additional 12 h under the same conditions, resulting in a total transfection time of 24 h. Cells were washed with PBS, trypsinized, and analyzed. Luciferase gene expression was quantified by using a commercial kit and a luminometer. Protein concentrations in the samples were analyzed by use of a bicinchoninic acid assay. Absorption was measured with a microplate reader at 570 nm and compared to a standard curve calibrated with BSA samples of known concentration.
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Full Papers Results are expressed as relative light units per milligram of cell protein lysate (RLU/mg protein).
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Manuscript received: April 22, 2015 Accepted article published: May 28, 2015 Final article published: June 11, 2015
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