Multivalent Dendrimer Vectors with DNA Intercalation Motifs for

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Multivalent Dendrimer Vectors with DNA Intercalation Motifs for Gene Delivery Pamela T. Wong, Kenny Tang, Alexa Coulter, Shengzhuang Tang, James R. Baker, and Seok Ki Choi Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/bm501169s • Publication Date (Web): 06 Oct 2014 Downloaded from http://pubs.acs.org on October 14, 2014

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Multivalent Dendrimer Vectors with DNA Intercalation Motifs for Gene Delivery Pamela T. Wong,1,2 Kenny Tang,1 Alexa Coulter,1 Shengzhuang Tang,1,2 James R. Baker, Jr.,1,2 and Seok Ki Choi1,2,* 1

Michigan Nanotechnology Institute for Medicine and Biological Sciences, 2Department of

Internal Medicine, University of Michigan Medical School, Ann Arbor, Michigan 48109, United States

*

To whom correspondence should be addressed,

Phone: (734) 615-0618; Fax: (734) 615-0621; Email: [email protected]

ACS Paragon Plus Environment

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Abstract Poly(amido amine) (PAMAM) dendrimers constitute an important class of nonviral, cationic vectors in gene delivery. Here we report on a new concept for dendrimer vector design based on the incorporation of dual binding motifs: DNA intercalation, and receptor recognition for targeted delivery. We prepared a series of dendrimer conjugates derived from a fifth generation (G5) PAMAM dendrimer, each conjugated with multiple folate (FA) or riboflavin (RF) ligands for cell receptor targeting, and/or with 3,8-diamino-6-phenylphenanthridinium (“DAPP”)derived ligands for anchoring a DNA payload. Polyplexes of each dendrimer with calf thymus dsDNA were made and characterized by surface plasmon resonance (SPR) spectroscopy, dynamic light scattering (DLS) and zeta potential measurement. These studies provided evidence supporting polyplex formation based on the observation of tight DNA-dendrimer adhesion, and changes in particle size and surface charge upon co-incubation. Further SPR studies to investigate the adhesion of the polyplex to a model surface immobilized with folate binding protein (FBP), demonstrated that the DNA payload has only a minimal effect on the receptor binding activity of the polyplex: KD = 0.22 nM for G5(FA)(DAPP) vs 0.98 nM for its polyplex. Finally, we performed in vitro transfection assays to determine the efficiency of conjugate mediated delivery of a luciferase-encoding plasmid into the KB cancer cell line, and showed that RF-conjugated dendrimers were one to two orders of magnitude more effective in enhancing luciferase gene transfection than a plasmid only control. In summary, this study serves as a proof of concept for DNA-ligand intercalation as a motif in the design of multivalent dendrimer vectors for targeted gene delivery. Key Words: PAMAM Dendrimer, DNA Intercalator, Riboflavin, Folic Acid, Multivalent Binding, Surface Plasmon Resonance Spectroscopy, Gene Transfection


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Introduction The design of efficient nonviral vectors for gene delivery and transfection requires consideration of numerous extracellular and intracellular aspects.1-4 Factors for vector design include the molecular mechanism for complexation that occurs between vectors and DNA. Complexation plays a crucial role in determining the efficiency of gene transfection as it underlies the biophysical and physicochemical properties of the complexes which dictate their modes of cellular entry and routes of endolysomal escape—a large barrier to gene delivery.1,4-6 Most of the current existing nonviral vectors are based on cationic polymers, lipids and peptides.1,4,7-23 The mechanism for the formation of lipoplexes and polyplexes made from these cationic vectors is primarily based on electrostatic interactions between the polycation and DNA polyanion.6,24 Despite the direct influence of complexation on transfection efficiency, other modes of DNA-vector association have been far less investigated with only a few reported including sequence-specific association,25 triple helix formation,26 and covalent attachment27-29 to DNA. In this study, we investigate a new mechanism of nonviral vector design with a surface receptor-targeting dendrimer nanoparticle conjugated with a multivalent array of ligands capable of DNA intercalation (Figure 1). Nucleic acid intercalation refers to a specific mechanism of binding in which a small molecule ligand forms π-π stacking interactions between adjacent base pairs of DNA and RNA.30,31 We were interested in applying this mechanism to the design of new nonviral vectors. Unlike ionic interactions,5 intercalation is ligand specific and relatively insensitive to pH variation,30,32,33 and thus allows for retention of the helical structure of DNA without leading to DNA aggregation or condensation during complexation.5,34 In addition, numerous types of high affinity intercalators are readily amenable to conjugation including ethidium (KD ≈ 10−5–10−6 M),30,32 acridine (KD ≈


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10−5 M),33 and 3,8-di-amino-6-phenylphenanthridine (DAPP; KD = 2.2 × 10-7 M).35 We hypothesize that these properties give intercalation several unique advantages that potentially address the current shortcomings in polyplex-based gene delivery. In this study, we investigated the design of nonviral vectors using a fifth generation (G5) PAMAM dendrimer36,37 as the scaffold for multivalent ligand design for DNA intercalation. As a prototype of synthetic dendritic polymers (diameter = 5.4 nm),36,37 the G5 dendrimer has been extensively investigated for targeted drug38-43 and gene9-12,15 delivery (Scheme 1). Our earlier studies showed that the predefined orientation of its terminal branches offers a platform ideal for tight multivalent binding to receptors on the cell surface.39,42,44-46 Thus, the G5 dendrimer has been established as a multifunctional platform for anticancer applications for targeting cancerspecific surface biomarkers.14,41,42,44,45,47-52 The current study uses two dendrimer types G5(FA)44,47 and G5(RF),

49,50

each conjugated

with folic acid (FA) or riboflavin (RF) as the cell targeting ligand, for the design of a new class of dendrimer vectors by integration of a DNA intercalation motif (Scheme 1). We first prepared and investigated a series of multivalent dendrimers designed with a DNA intercalation motif for their ability to form polyplexes. Second, we investigated whether such polyplexes could bind to their targeted receptors as tightly as free dendrimer using a folate receptor (FAR) model system. High avidity binding of the polyplex to the receptor is critical for transfection efficiency as it enables cellular uptake of the polyplexes via receptor-based endocytosis.53,54 Lastly, we investigated whether polyplexes of plasmid DNA (pDNA) containing a luciferase gene enhanced expression of this reporter gene in KB tumor cells in vitro. The results of these experiments strongly suggest that DNA intercalation is an effective new mechanism for nonviral vector


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design that should be taken into consideration in the development of cancer targeted gene delivery systems. DNA Plasmid-Dendrimer Complex

dsDNA d ~ 2 nm

Luciferase

d~ 5 nm Dendrimer DNA intercalator Targeting ligand

Receptor

Figure 1. A schematic illustrating the proposed concept for multivalent dendrimer vectors with DNA intercalation motifs for targeted delivery of DNA payloads to a cancer cell. The dendrimer conjugate presents a multivalent array of two functionally orthogonal ligands, one for anchoring DNA payloads and the other for targeting cancer cell associated receptors. Experimental Section Materials and analytical methods (NMR spectroscopy, MALDI-TOF mass, HPLC, GPC) are described in details in the Supporting Information and references cited therein. Synthesis of G5 PAMAM Dendrimer Conjugates 3–7 (Scheme 1; see Supporting Information). Folic acid (21.6 mg, 49 µmol) and 1 RF-CO2H50 (24 mg, 49 µmol) was activated each separately to its 1-hydroxybenzotriazolyl (HOBt) ester in DMSO (1.0 mL) by using PyBOP ((benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate, 60 µmol). 2 DAPPCO2H55 (32.3 mg, 56.5 µmol) was similarly activated to its HOBt ester in DMSO (1.5 mL). Each activated solution (FA-HOBT ester, RF-HOBT ester, DAPP-HOBt ester) reacted singly or in


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combination (Scheme 1) with G5 PAMAM dendrimer (Mr = 27,600) dissolved in MeOH, and unreacted primary amines of the dendrimer were neutralized by treatment with acetic anhydride (60 molar equiv). Each dendrimer conjugate was purified by membrane dialysis tubing (MWCO 10 kDa), and characterized by analytical UPLC, UV–vis spectrometry, matrix assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry, and

1

H NMR

spectroscopy. Valency of each ligand attached was determined on a mean basis by a UV–vis method using a calibration curve prepared for each ligand. Full details for each step, dendrimer purification and characterization are described in Supporting Information. Surface Plasmon Resonance (SPR) Spectroscopy. SPR experiments were performed on a Biacore® X instrument (Pharmacia Biosensor, AB) using a CM5 sensor chip. FBP or dendrimer immobilization and SPR experiments were performed as described in Supporting Information.44,45,47 SPR data were analyzed by global fitting to a Langmuir model.56 Full details for this SPR study are described in Supporting Information. Dynamic Light Scattering and Zeta Potential. The size and charge distribution of dendrimer conjugates and their DNA polyplexes were determined by dynamic light scattering (DLS) and zeta potential, each measured at room temp on a Zetasizer Nano ZS (Malvern).57 Briefly, 0.9 mg/mL of each dendrimer (~10 nM) was incubated with 0, 0.05, 0.1, or 0.2 mg/mL of calf thymus DNA in 1 mM HEPES pH 7 for 10 m at room temp. Particle size and zeta potential measurements were performed consecutively. Cell Culture. KB cells were maintained in RPMI 1640 medium with no folic acid (Life Technologies) with 10% heat-inactivated fetal bovine serum (FBS), 100 IU penicillin, and 100 µg/mL streptomycin. All studies were performed with media lacking folic acid.


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Luciferase Transfection. KB cells were seeded overnight in 24 well tissue culture plates in media containing FBS. The transfected luciferase gene was located on a pGL4.50 plasmid obtained from Promega (Madison, WI) (pLuc). Dendrimer stocks were dissolved in water, and sonicated briefly in a water bath sonicator to disperse the particles. Dendrimer/pLuc polyplexes were formed by mixing dendrimer and pLuc at a higher concentration for 15 min in FBS-free media at room temp (22 µg/mL dendrimer with 11 µg/mL pLuc for the 2:1 ratio, and 11 µg/mL dendrimer with 11 µg/mL pLuc for the 1:1 ratio) to allow for complex formation. Cells were rinsed twice in media without FBS. The dendrimer/pLuc mixture was then diluted in FBS-free media such that a total of 1µg pLuc was added to each well, mixed with 1 µg or 2 µg of dendrimer (1:1, and 2:1 dendrimer:pDNA ratios, respectively) in 200 µL. Cells were incubated with dendrimer/pLuc for 4 h at 37̊C. One mL of media with FBS was added and the cells were incubated for an additional 48 h at 37̊C. Cells were scraped off the plate, and placed in a centrifuge tube along with the supernatant. Cells were spun at 8000 rpm for 5 min in a microfuge, and the cells were washed twice with cold PBS. To lyse the cells, 200 µL of reporter lysis buffer (Promega) was added, and the cells were vortexed vigorously for 15 s, and frozen at −20̊C to facilitate lysis. The cells were thawed, and spun down at 13,000 rpm for 30 s. The supernatant lysate was collected. The total protein concentration of each lysate was measured using a BCA assay (ThermoFisher). Luciferase activity was measured using the Promega Luciferase Assay System according to the manufacturer’s protocol. Briefly, 20 µL of lysate was added per well to a 96 well plate, and the luminometer (Centro XS3 LB 960, Berthold Technologies) was set to inject 100 µL of luciferase substrate per well with a 10 s read time and 2 s delay between readings. Luciferase activity is expressed as relative light units (RLU) per µg of total protein.


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Confocal Microscopy. KB cells were seeded overnight on chambered coverglass slides at 1x105 cells per well in riboflavin free media with FBS. Dendrimer/pLuciferase polyplexes were formed as described for the transfection assay in FBS-free media. Cells were rinsed twice with FBS free media, and 0.005 µg/mL or 0.01 µg/mL dendrimer complexed with 0.005 µg/mL pDNA was added to the cells in 200 µL of FBS-free media (1 µg or 2 µg dendrimer with 1 µg pDNA total), and incubated at 37̊C for 4 h. 600 µL of fresh media containing 10% FBS was then added and the cells were incubated for 24 h. Cells were rinsed with PBS, fixed in 4% paraformaldehyde for 10 min, and dried before Prolong Gold was added. Cells were imaged with a Leica DCS 480 epifluorescence microscope and analyzed with LAS software (Leica Microsystems). Scheme 1. Synthesis of fifth generation (G5) poly(amido amine) (PAMAM) dendrimer conjugates 3–7a,b


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a

Each dendrimer nanoparticle shows only a fraction of its internal and external branches for clarity (terminal branches = 128 (theor);37 114 (exptl)58,59). b Reagents and conditions: i) FAHOBt ester (13 equiv), MeOH, rt; ii) Ac2O (60 equiv), Et3N; iii) 2 DAPP-HOBt ester (10 equiv), MeOH, rt; iv) FA-HOBt ester (13 equiv), 2 DAPP-HOBt ester (10 equiv), MeOH, rt; v) 1 RFHOBt ester (13 equiv), MeOH, rt; vi) 1 RF-HOBt ester (13 equiv), 2 DAPP-HOBt ester (10 equiv), MeOH, rt. abbreviation: HOBt = 1-hydroxybenzotriazole; rt = room temp Results and Discussion Design of Dendrimer Vectors. The G5 PAMAM dendrimer,37,60 G5-(NH2)n was used as the nanometer-sized multivalent scaffold for ligand conjugation. The periphery of this dendrimer provides a large number of primary amine branches (ntheor = 128),36,37 and each of its terminal branches is available for covalent conjugation with cancer targeting ligands and DNA intercalators. Our design approach for cancer cell-targeting dendrimer vectors involves


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conjugation with a combination of two functionally orthogonal ligands or a dual-functional (targeting) ligand (Scheme 1): i) folic acid (FA) or riboflavin (RF) as the cancer receptor targeting ligands; ii) 3,8-di-amino-6-phenylphenanthridine (DAPP) as a DNA intercalator for anchoring DNA payloads. FA (vitamin B9)41,43,45 serves as a standard ligand of choice employed in cancer targeted delivery of genes and therapeutic agents due to its specific affinity to the folate receptor overexpressed on tumor cells (KD = 0.4 nM),61 and due to the well-established mechanism of receptor-mediated endocytosis of polymers and nanomaterials conjugated with FA.62 Here we also employed RF (vitamin B2)49,50,53,63 for cancer targeting as has been investigated in KB cells48,64 given that it binds with high affinity (KD = 5 nM64) to riboflavin receptors (RFR) on the cell surface—another family of tumor biomarkers over-expressed in certain human tumors (breast, prostate).63,65 DAPP was selected as the small molecule ligand for anchoring pDNA to the dendrimer, due to its ability to tightly bind dsDNA (KD = 2.2 × 10-7 M35) through non-covalent π-π stacking interactions of its fused heteroaromatic system with adjacent DNA base pairs (Scheme 1).34,66 It is noteworthy that RF can play a dual role in this design. In addition to its binding affinity to RFR on the cell surface, it is also known to bind nucleotides through intercalation of its flat flavin aromatic ring structure with base pairs,67 and perhaps though less relevant through binding to abasic (AP) sites (KD = 5.5 × 10−7 M)68,69 that are known to arise randomly in plasmid DNAs with a significant frequency of ~100,000 AP sites per cell.70 By consideration of all these roles played by these three ligands, we designed a series of five G5 conjugates by conjugation with a single ligand or two ligands as described below. Synthesis of Dendrimer Vectors. Unlike FA which has two carboxylic acids (α, γ) on its glutamate residue, each of the two ligands, RF and DAPP, lacks a carboxylic acid group as a chemical handle needed for amide conjugation with the terminal amine of the dendrimer, and


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thus required structural derivatization (Scheme 1). RF was derivatized to 1 RF-CO2H at its (D)ribose unit at the N-10 position as described earlier in the design of its PAMAM dendrimer conjugates.48,50 The glutaric acid (GA) spacer tethered to the (D)-ribose unit is expected to be exposed to the aqueous medium such that the entire RF molecule can participate in forming a complex with the RFR.71 For DAPP derivatization, we performed an N-alkylation reaction at the N5-position with 4-(bromomethyl)benzoate as previously reported in the literature55 as this N5modification55,72 allows for retention of the DNA binding affinity of the resulting derivatives including 2 DAPP-CO2H. We next preactivated each of these ligands through converting them into amine-reactive esters by

treatment

with

benzotriazol-1-yl-oxytripyrrolidinophosphonium

hexafluorophosphate

(PyBOP) and N-hydroxybenzotriazole (HOBt). Each activated ester was subsequently reacted with G5-(NH2)114 individually to yield 3 G5(FA)n, 4 G5(DAPP)m and 6 G5(RF)n’ or in combination with another orthogonal ligand to

yield 5 G5(FA)n(DAPP)m and 7

G5(RF)n’(DAPP)m’. The amount of each ligand added for dendrimer conjugation varied from 10– 13 molar equiv since our earlier studies suggest a sufficient ligand valency for multivalent receptor binding as n ≈ 3–10.40,45,47 After this coupling step, most of the unreacted primary amines on the dendrimer surface were neutralized to N-Ac amide groups by treatment with acetic anhydride (60 molar equiv). Each of the ligand-conjugated dendrimers 3–7 prepared by the stochastic method above was purified by dialysis in a membrane tubing (molecular weight cut off = MWCO = 10 kDa), and small molecule-based impurities including free ligand molecules and reagents were not detectable by analytical HPLC (polymer purity ≥95%; Figure S1, Supporting Information) after the dialysis step. Further characterization of each conjugate was performed by standard


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analytical methods including matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), gel permeation chromatography (GPC),

1

H NMR

spectroscopy, and UV–vis spectrometry. As summarized in Table S1, values of Mr determined by MALDI-TOF are comparable among all the conjugates and used for calculating the concentrations of dendrimer stock solutions. In addition to MALDI-TOF, the weight-average molecular weights (Mw) of conjugates 4–7 were determined by GPC. These two sets of independently measured data show generally low to moderate deviations (= [(Mr−Mw)/Mr] × 100%) lying within ±0.4 to 23%. In addition, GPC measurement provided a distribution in the molecular weights (polydispersity index = PDI = Mw/Mn ≈ 1.1–1.5) of these ligand-modified dendrimers. Two conjugates 4 and 7 showed wider GPC distributions, largely due to the presence of small fractions of faster-eluting (larger) polymer species, than the other conjugates, 3 and 6 (Figure S1B, Supporting Information), suggesting a possible greater contribution of the DAPP ligand to the higher heterogeneity of these conjugates. 1H NMR spectral data indicate the presence of ligand-associated protons in each spectrum (Figure S2, Supporting Information). UV–vis absorption features are also strongly consistent with the attachment of each ligand (Figure S3, Supporting Information): 3 (FA): λmax = 350 nm (ε = 62,600 M−1cm−1); 4 (DAPP): λmax = 350 nm (ε = 99,980 M−1cm−1); 6 (RF): λmax = 448 nm (ε = 5,900 M−1cm−1). Ligand valency (Table S1) was determined for each dendrimer conjugate on a mean basis by the analysis of the UV–vis absorptivity as described in detail in the Experimental section. Design of Dendrimer-immobilized Biosensor Chips. We employed SPR spectroscopy to investigate whether dsDNA binds to dendrimers functionalized with the DNA intercalator as


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prepared above. SPR is a biophysical method that measures the binding kinetics, and thus affinity of an analyte to a model surface, and has been used for studying various biological interactions including those between multivalent dendrimers and receptors44-47 and nucleic acids and intercalators.73,74 Earlier SPR studies of nucleic acid-intercalator interactions utilized a model surface prepared by immobilization of either model nucleotides73 or intercalators.74 In this study, we preferred the latter approach because a model surface immobilized with dendrimer intercalators allows the study of native DNA molecules without any chemical modifications. Furthermore, collection of SPR data requires surface regeneration after each analyte injection by treatment with a highly acidic buffer solution (10 mM glycine–HCl, pH 1.5) such that the acidlabile ribose units of the DNA strand immobilized to the surface may undergo partial or full cleavage over the course of the experiments. SPR Study of DNA-Dendrimer Complexation. We designed a CM5 sensor chip such that flow cell 1 was immobilized with 4 G5(DAPP)5.4 as the DNA-binding dendrimer intercalator conjugate, while flow cell 2 was treated in the same manner without the dendrimer to serve as a reference surface (Figure S4A, Supporting Information). As anticipated, tight and specific adhesion of dsDNA was observed on flow cell 1 in a dose-dependent manner (Figure 2B, C). The association was permanent, as it showed extremely slow dissociation (kd < 8.8 (±5.8) × 10-6 s-1) such that almost all of adsorbed DNA molecules remain still bound until the end of the dissociation period.


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Figure 2. SPR experiments for DNA-dendrimer interaction on the surface. (A) Design of a sensor chip in which flow cell 1 is immobilized with 4 G5(DAPP)5.4 and flow cell 2 serves as a reference surface. Concentration-dependent binding sensorgrams of dsDNA (calf thymus) alone in flow cell 1 (B) and flow cell 2 (C). Concentration-dependent binding sensorgrams of polyplexes made of dsDNA with 4 G5(DAPP)5.4 (D) and 6 G5(RF)4.9 (E), each prepared at a 1:1 ratio (w/w). Each sensorgram shown (D, E) is corrected against the reference sensorgram: ∆ Response Unit (RU) = RU (Fc1) – RU (Fc2). [dsDNA]injected = [Dendrimer]injected = 8.0 µg/mL (a), 4.0 (b), 2.0 (c), 1.0 (d), 0.5 (e). ∆RUmax refers to the maximal ∆RU reached before dissociation begins as illustrated in (D). We tested whether such DNA adhesion is specific to the immobilized dendrimer 4 on the surface of the chip by using a competitive inhibition method in which free 4 was mixed with the


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DNA (1:1, w/w), and this preformed 4-DNA polyplex was injected. While 4 alone did not show any specific binding to the G5(DAPP)5.4 immobilized chip (Figure S5B, Supporting Information), its complexation with DNA led to strong competitive inhibition of the DNA adhesion to the dendrimer surface in a dose-dependent manner, which is supportive of binding specificity (Figure 2D). We next tested 6 G5(RF)4.9 for its ability to form a complex with dsDNA because RF binds nucleotides via intercalation and H-bonding interactions with its flavin ring.6769

The preformed DNA polyplex made with 6 abolished DNA adhesion to flow cell 1 (Figure

2E), supporting DNA intercalation by RF. Such competitive inhibition was also observed by DNA pre-complexation with 5 G5(FA)8.6(DAPP)5.4 and 7 G5(RF)4.9(DAPP)6.9 (Figure S5D, F, Supporting Information) which also present a multivalent array of the RF and/or DAPP ligand.


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Figure 3. Plots of ∆RUmax values measured in the sensorgrams of various polyplexes (A), and corresponding decreases (%) in ∆RUmax relative to DNA alone (B). All SPR sensorgrams analyzed for preparing these plots are shown in Figure 2 (and Figure S5). ∆RUmax is defined in Figure 2D and its change (%) is calculated at each identical concentration as follows: ([∆RUmax, DNA

− ∆RUmax,

polyplex]/∆RUmax, DNA)

× 100%. As described, each polyplex was preformed by

mixing calf thymus dsDNA with 4 G5(DAPP)5.4, 5 G5(FA)8.6(DAPP)5.4, 6 G5(RF)4.9, or 7 G5(RF)4.9(DAPP)6.9 at a 1:1 ratio (w/w) at the given concentration (0.5–8.0 µg/mL) before injection on to the 4 G5(DAPP)5.4-immobilized sensor chip. (C) A plot of the dissociation rate constant (kd) determined for the desorption of DNA alone compared to that of each polyplex bound to the chip surface.


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Analysis of dsDNA Adsorption to 4-immobilized Chip. Quantitative comparisons of the competitive inhibition of DNA binding to the chip by each dendrimer type are summarized in Figure 3. We used the value of ∆RUmax (as defined in Figure 2D) as the metric because it is proportional to the mass of DNA or polyplex adsorbed to the surface. A plot of ∆RUmax values as calculated from the sensorgrams of these polyplexes was prepared along with a corresponding plot comparing the decreases in ∆RUmax (%) relative to the ∆RUmax for DNA alone (([∆RUmax, DNA

− ∆RUmax, polyplex]/∆RUmax, DNA) × 100%). These plots indicate that this competitive inhibition

is more effective with 4 and 7 than with 5 or 6. We believe that the effectiveness of such competitive inhibition is possibly related to the efficiency of polyplex formation and determined by design factors such as the type of intercalating ligand and valency employed. We also determined the rate constant of dissociation (kd) for free dsDNA or polyplexes bound to the 4-immobilzied sensor chip surface (Figure 3C). While an equilibrium dissociation constant (KD) is also considered useful for evaluation of the avidity of each dendrimer to dsDNA, its analysis is not applicable here as the Mr of the calf thymus dsDNA is unknown due to the heterogeneity of this DNA. As stated earlier, dsDNA alone showed very slow dissociation from the 4-immobilized chip (kd < 8.8 (±5.8) × 10-6 s-1). In contrast, dsDNA bound in certain polyplexes showed much faster dissociation rates that are three to four orders of magnitude greater than dsDNA alone, as seen for polyplex 4 (kd = 5.0 (±4.1) × 10-2 s-1) and polyplex 7 (kd = 1.1 (±0.43) × 10-2 s-1). Two other polyplexes also showed faster dissociation than DNA alone by one to two orders of magnitude: polyplex 6 (kd = 2.4 (±1.2) × 10-3 s-1) and polyplex 5 (kd < 6.7 × 10-5 s-1). We believe that the decrease in DNA adsorption (∆RUmax) and the increase in dissociation rate are strongly indicative of the relative ability of each of the dendrimer conjugates


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4–7 to form polyplexes with dsDNA, which subsequently leads to the competitive inhibition of dsDNA adsorption to the chip immobilized with the dendrimer 4. Dynamic Light Scattering and Zeta Potential Measurement of DNA-Dendrimer Complexes. To further characterize the properties of the dendrimer-DNA complexes in solution, the particle size and zeta potential (ZP) of dendrimer polyplexes with increasing concentrations of calf thymus dsDNA were measured following a previously published protocol (Figure 4).57 Dynamic light scattering (DLS) was used to measure the particle size of the polyplexes. Distributions of particle diameter (Zave) for dendrimer alone and dendrimer with 0.1 mg/mL of calf thymus DNA are shown in Figure 4A. In both cases, particle sizes for the four dendrimers were relatively similar, and overall had unimodal size distributions. The Zave for 4 G5(DAPP)5.4, 5 G5(FA)8.6(DAPP)5.4, 6 G5(RF)4.9, and 7 G5(RF)4.9(DAPP)6.9 alone were 249.6, 118.5, 198.3, and 144.1 nm, respectively (Table S2, Supporting Information).


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Figure 4. Size and charge properties of dendrimer-DNA polyplexes characterized by dynamic light scattering (DLS) and zeta potential (ZP) measurements. (A) DLS size distribution plots of 0.9 mg/mL dendrimer alone (4 G5(DAPP)5.4, 5 G5(FA)8.6(DAPP)5.4, 6 G5(RF)4.9, and 7 G5(RF)4.9(DAPP)6.9), or with 0.1 mg/mL calf thymus DNA. Particle size is shown as diameter


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(Zave). (B) The ZP values of dendrimer alone (0.9 mg/mL) and its polyplexes as a function of calf thymus DNA concentration as measured in 1 mM HEPES pH 7. Addition of dsDNA to the dendrimers altered the size distributions for all four conjugates. Upon addition of dsDNA, 4, 5, and 7 all showed the appearance of a small peak corresponding to a very large particle size >2000 nm, with 4, and 5 containing particles beyond the size detection limit of the instrument (Figure 4). These results clearly demonstrate an interaction between the dsDNA and dendrimer, as this complex is larger than both dendrimer alone and dsDNA alone. Furthermore, the predominant peak for all four dendrimers showed both a shift towards slightly larger particle sizes, and also a broadening in the peak. In other words, particles with slightly larger diameters were observed for all four dendrimers, as well as particles with slightly smaller hydrodynamic diameters upon addition of dsDNA in comparison to dendrimer alone. While the smaller particles fall within the range of the dsDNA alone, it is unlikely due to free DNA, as the size distribution of DNA alone is highly polydisperse, and at this low DNA-to-dendrimer ratio (= 0.11 w/w), the contribution of DNA to the light scattering signal is minimal (Figure 4A, Table S2, Supporting Information). The dendrimer alone is expected to be only around 10 nm in diameter (at pH 2.7, as determined by gel permeation chromatography). However, the larger hydrodynamic diameter 100–250 nm observed for the dendrimer alone could reflect the presence of loosely associated dendrimer-dendrimer complexes at the neutral pH used for the DLS measurements, which then dissociate to smaller units upon coating of the dendrimer with dsDNA. Zave values for the dendrimer-DNA complexes are summarized in Figure 4B (Table S2, Supporting Information), including those at the intermediate DNA concentration of 0.05 mg/mL, and show DNA concentration-dependent changes in average particle size. Thus, while the


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dendrimer conjugates alone are relatively homogenous in size as indicated by narrow PDI values by GPC (1.066–1.501; Table S1), polyplexes with dsDNA show greater heterogeneity in particle size, with some formation of very large dendrimer-DNA complexes. This observation is based on complexation with calf thymus DNA, but is in general agreement with other studies in which greater size heterogeneity for polyplexes was observed in comparison to the parent dendrimers.5,75,76 The ZPs for 4 G5(DAPP)5.4, 5 G5(FA)8.6(DAPP)5.4, 6 G5(RF)4.9, and 7 G5(RF)4.9(DAPP)6.9 were 54.6 ± 9.4, 48.4 ± 8.1, 46.7 ± 5.1, and 52.1 ± 12.7 mV, respectively. Thus the surface branches of the dendrimer conjugates are all partly cationic due to the presence of unmodified primary amines (~40–45/particle). To measure polyplex formation, the ZPs of the dendrimer conjugates in the presence of calf thymus dsDNA at two different dendrimer:dsDNA ratios (1:18 and 1:9 (0.05 and 0.1 mg/mL dsDNA, respectively)) were measured. Addition of calf thymus dsDNA, which is highly negatively charged (ZP = −78.1 ± 3.9 mV) resulted in a significant drop in the dendrimer surface charge, in a DNA concentration-dependent manner for all four conjugates. At the highest dsDNA concentration (0.1 mg/mL), the ∆ZP (ZPfinal − ZPinit) for 4, 5, 6, and 7 were 16.2, 8.8, 15.7, and 12 mV, respectively (Table S2, Supporting Information). Comparison of the ∆ZP (ZPfinal − ZPinit) values for 4, 5, 6, and 7 (Table S2, Supporting Information),showed that at the lower dsDNA concentration (0.05 mg/mL), the largest ∆ZP was observed for 6, possibly reflecting a difference in the association of 6 with DNA as compared to the other dendrimers. At these dendrimer:DNA ratios, the dendrimers retain a significant proportion of their cationic charge. These findings were consistent with the implications of the SPR results, supporting dendrimer association and surface coating with dsDNA.


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SPR Study of Polyplex Avidity. The results observed above for SPR, size and ZP measurements support the formation of polyplexes through DNA intercalation. We were thus interested in determining whether the DNA payload interferes with the binding of the dendrimer carrier to its targeted receptors on the surface. We hypothesize that retaining such activity for receptor binding opens an important route for the cellular uptake of these polyplexes via receptor-based endocytosis, and constitutes one of the critical factors that dictate the overall efficiency of gene delivery. SPR experiments were designed to compare the receptor binding avidity of the dendrimers alone and their corresponding polyplexes. For this SPR study, FAR or RFR can be considered for studying dendrimers 4–7. However, we tested our hypothesis by employing only FAR as methods for receptor immobilization on the chip surface are well established with the use of folate binding protein (FBP) as a model protein,44-47 whereas no method exists for RFR. The FBP used here is commonly used as a surrogate protein for FAR in studies to characterize ligand binding, as FBP is more soluble than the membrane receptor, and has extremely high homology to the FAR which is overexpressed on the cell surface of certain tumor cell types.42-44, 68 A model surface for FAR44,45,47 was designed by immobilization of FBP (bovine) on the surface of a CM5 sensor chip through amide coupling by an EDC/NHS-based method as described elsewhere.44,45,47 The protein immobilization led to a surface density of 13.1 FBP ng/mm2 (Figure S4B, Supporting Information; 1000 RU = 1 ng protein/mm2), which is equivalent to a number density of 2.6 × 1011 FBP molecules per mm2. We estimate that such density is comparable to the level of FAR present on several malignant cells which express the receptor at levels 10- to 20-fold higher than normal cells.77,78


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We assessed this FBP chip for its binding affinity to the FA ligand by acquiring dosedependent SPR sensorgrams (Figure S4C, Supporting Information). The equilibrium dissociation constant KD (kd/ka) determined for FA (KD = 6 × 10-6 M; Table S3) was consistent with those values reported in the literature (KD = 2 × 10-6 M, 5 (±3) × 10-6 M).44,45 We performed SPR binding

studies

first

for

FA-conjugated

dendrimers

alone,

3

G5(FA)8.6

and

5

G5(FA)8.6(DAPP)5.4, each at six different concentrations (Figure 5B, D). The SPR binding kinetics of two polyplex types, each made with calf thymus dsDNA in complex with 3 or 5, to the same FBP sensor chip were then measured (Figure 5C, E).


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Figure 5. (A) Design of a sensor chip composed of folate binding protein (FBP) immobilized in flow cell 1 and a reference surface in flow cell2. (B to E) Concentration-dependent SPR sensorgrams of dendrimer alone 3 G5(FA)8.6 (B), 5 G5(FA)8.6(DAPP)5.4 (D) or each of their polyplexes with calf thymus dsDNA (C, E). [dendrimer] = 3.2 µg/mL (a), 1.6 (b), 0.78 (c), 0.39 (d), 0.2 (e), 0.1 (f). [dsDNA] = 0.25 µg/mL. Dotted lines represent simulated global fits. In SPR binding studies for 3 G5(FA)8.6 and 5 G5(FA)8.6(DAPP)5.4, each conjugate showed dose-dependent adsorption to the FBP surface (flow cell 1) with some level of nonspecific binding to the reference surface (flow cell 2),attributable in part to a bulk effect of the dendrimer solution (Figure S6, Supporting Information). In contrast, Ac-G5, a control dendrimer modified with acetamide (Ac) only, failed to show any level of adsorption to the FBP surface (not shown),79 suggesting the binding specificity of the FA-conjugated dendrimers. The sensorgrams from conjugates 3 and 5 show markedly slow dissociation compared to the rapid dissociation displayed by free FA (Figure S4C, Supporting Information). Such slow rates of dissociation were observed in our earlier studies,39,45,47 and are characteristic of other multivalent systems.80,81 The kinetic parameters of multivalent binding by each conjugate were quantitatively analyzed by global fitting56 to a Langmuir binding isotherm as described in the Experimental section, and rate constants (ka, kd) and KD values were extracted from best fits as summarized in Table S3. These values suggest that each FA-conjugated dendrimer binds very tightly to the FBP surface with subnanomolar dissociation constants (KD ~ 10−10 M), an enhancement in binding avidity of approximately four orders of magnitude relative to the monovalent affinity of free FA (KD ~ 10−6 M). Such high avidity is consistent with earlier results observed for other FAconjugated multivalent dendrimers,44 and also confirms the choice of this ligand valency (mean 8.6 FA per dendrimer) as optimal for cancer targeted delivery applications.


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In the SPR analysis of the binding kinetics of two polyplex types (Figure 5C, E), each of these polyplexes showed qualitatively similar binding features as those observed for the corresponding dendrimer alone, such as slow dissociation from the FBP-immobilized chip (above). Quantitative analysis of these sensorgrams showed differences in the kinetic features and dissociation constants of these two polyplexes (Table S3). The polyplex made with 3 binds to the FBP surface as tightly as dendrimer 3 G5(FA)8.6 alone, and its absolute mass of adsorption remains almost the same (96%). In contrast, the other polyplex made with 5 G5(FA)8.6(DAPP)5.4 showed a lowered mass of adsorption (86%) as well as a factor of five decrease in the avidity of the bound polyplexes. This difference lies in the presence of the DAPP ligand in dendrimer 5, which plays a role in dendrimer-DNA complexation as a DNA intercalator. Thus, the data suggest that the presence of DNA bound to dendrimer 5 may exert some level of steric hindrance to FA ligands on the dendrimer, and result in a decrease in FA receptor binding and dendrimer avidity as well. However, the SPR results illustrate that such a decrease is not significant and the resulting polyplex still binds tightly to the FBP surface with nanomolar avidity. In Vitro Luciferase Transfection. To determine the effectiveness of the polyplexes in facilitating gene transfection, we evaluated the transfection of pDNA containing a luciferase gene (pLuc) in KB cells. As KB cells overexpress both FAR and RFR,48,53,82 the effects of targeted delivery could be studied. Transfections were performed with polyplexes composed of variable amounts of dendrimer and pLuc pDNA at multiple dendrimer:pDNA ratios (D/P) to optimize the assay conditions. The commercial transfection reagent, TransFast was used as a positive control. One µg of plasmid with D/P ratios of 2:1 and 1:1 (w/w) with a 4 h transfection time was identified as the optimal condition for the assay (Figure S7A, Supporting Information). Transfection of pLuc was also evaluated for polyplexes of two selected dendrimer controls:


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G5(GA)59 (GA = glutaric acid), and G5-NH2. The negatively-charged G5(GA) was ineffective as a transfection agent, while G5-NH2 significantly enhanced the transfection efficiency relative to that of DNA alone, which is consistent with what has been previously reported (Figure S7B, Supporting Information).9 The transfection activities of polyplexes made with dendrimer conjugates 3 G5(FA)8.6, 4 G5(DAPP)5.4, 5 G5(FA)8.6(DAPP)5.4, 6 G5(RF)4.9 and 7 G5(RF)4.9(DAPP)6.9 were observed in the absence (Figure 6) or presence (Figure S8) of FBS. The presence of FBS during transfection had only minor effects on the magnitude of the transfection efficiency in this assay (Figure S8), suggesting the stability of these polyplexes in serum and the lack of undesirable aggregation induced by the serum proteins.


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(A) 1:1 (w/w; n = 7) p*

Multivalent Dendrimer Vectors with DNA Intercalation Motifs for

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