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CD44-Targeted Hyaluronic Acid-Coated RedoxResponsive Hyperbranched Poly (amido amine)/Plasmid DNA Ternary Nanoassemblies for Efficient Gene Delivery Jijin Gu, Xinyi Chen, Xiaoqing Ren, Xiulei Zhang, Xiaoling Fang, and Xianyi Sha Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.6b00240 • Publication Date (Web): 16 Jun 2016 Downloaded from http://pubs.acs.org on June 19, 2016

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Bioconjugate Chemistry

CD44-Targeted Hyaluronic Acid-Coated Redox-Responsive Hyperbranched Poly (amido amine)/Plasmid DNA Ternary Nanoassemblies for Efficient Gene Delivery Jijin Gu †, ‡, Xinyi Chen †, Xiaoqing Ren †, Xiulei Zhang †, Xiaoling Fang †, Xianyi Sha*, †

†

Key Laboratory of Smart Drug Delivery (Fudan University), Ministry of Education1，Department of

Pharmaceutics, School of Pharmacy, Fudan University, 826 Zhangheng Road, Shanghai 201203, PR China ‡

Laboratory for Drug Delivery and Biomaterials, Faculty of Pharmacy, University of Manitoba, 750

McDermot Ave, Winnipeg, Manitoba, R3E 0T5, Canada

* Corresponding authors. Tel.: +86-21-51980071; Fax: +86-21-51980072. E-mail addresses: [email protected]

ACS Paragon Plus Environment


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ABSTRACT Hyaluronic acid (HA), which can specifically bind to CD44 receptor, is a specific ligand for targeting to CD44-overexpressing cancer cells. The current study aimed to develop ternary nanoassemblies based on HA-coating for targeted gene delivery to CD44-positive tumors. A novel reducible hyperbranched poly (amido amine) (RHB) was assembled with plasmid DNA (pDNA) to form RHB/pDNA nanoassemblies. HA/RHB/pDNA nanoassemblies were fabricated by coating HA on the surface of RHB/pDNA nanoassemblies core through electrostatic interaction. After optimization, HA/RHB/pDNA nanoassemblies were spherical, core-shell nanoparticles with nano-size (187.6 ± 11.4 nm) and negative charge (-9.1 ± 0.3 mV). The ternary nanoassemblies could efficiently protect the condensed pDNA from enzymatic degradation by DNase I, and HA could significantly improve the stability of nanoassemblies in the sodium heparin solution or serum in vitro. As expected, HA significantly decreased the cytotoxicity of RHB/pDNA nanoassemblies due to the negative surface charges. Moreover, it revealed that HA/RHB/pDNA nanoassemblies showed higher transfection activity than RHB/pDNA nanoassemblies in B16F10 cells, especially in the presence of serum in vitro. Because of the active recognition between HA and CD44 receptor, there was significantly different transfection efficiency between B16F10 (CD44+) and NIH3T3 (CD44-) cells after treated with HA/RHB/pDNA nanoassemblies. In addition, the cellular targeting and transfection activity of HA/RHB/pDNA nanoassemblies were further evaluated in vivo. The results indicated that the interaction between HA and CD44 receptor dramatically improved the accumulation of HA/RHB/pDNA nanoassemblies in CD44-positive tumor, leading to higher gene expression than RHB/pDNA nanoassemblies. Therefore, HA/RHB/pDNA ternary nanoassemblies may be a potential gene vector for delivery of therapeutic genes to treat CD44-overexpressing tumors in vivo. Keywords：


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CD44 targeting Hyaluronic acid Reducible hyperbranched poly (amido amine) Self-assembling Gene delivery

INTRODUCTION Cationic polymers-based polyplexes have been widely used as gene delivery vectors, because of their relatively superior transfection efficiency in vitro due to the efficient pDNA condensation and proton sponge effect.1,

2

However, their application in vivo has largely been limited by the

disadvantages including high cytotoxicity, lack of specificity, aggregation in blood, and unknown risk of long-term effects after accumulation.3-5 Thus, some novel and versatile polycations with high gene transfection activity are desirable to develop safe and efficient non-viral vectors for gene delivery. Reducible hyperbranched poly (amido amine)s (RHB) containing bioreducible disulfide linkages in the main chain have shown to be as a class of promising polymers for biomedical application, due to their biodegradability, low cytotoxicity, and high transfection efficiency.6 Disulfide bonds present in the structure of RHB are known to be stable in the blood and easily cleaved by glutathione (GSH) in the reducing intracellular environment. The intracellular cleavage of disulfide bonds in RHB is mostly mediated by thiol/disulfide exchange reaction with small redox molecules like GSH; either alone or with the help of redox enzymes. RHB is degraded into low molecular weight fragments inside the cells along with the reduction of disulfide linkages, resulting in the release of the entrapped pDNA. Hence, the reduction reaction accelerates the disassembly of the polyelectrolyte polycation/nucleic acid polyplexes and the release of pDNA, which is believed to decrease the cytotoxicity of polycations and



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to increase intracellular bioavailability of the delivered nucleic acids. However, most of the investigations have been focused on the transfection of RHB in vitro, and the success for in vivo application is still limited. The main potential obstacles of the in vivo application of RHB are positively charged surface characteristics and nonspecific delivery. The positive surface charge of RHB/nucleic acid polyplexes might lead to low stability in serum, erythrocytes aggregation, and short circulation in vivo. It has been recognized that introducing polyanions into polycations-based gene carriers by physical electrostatic coating can provide progressive advantages. The polyanion/polycation/pDNA ternary nanoassemblies based on electrostatic coating have attracted considerable attention as a promising gene delivery system.7 Various electrostatic coating biomaterials including poly (g-glutamic acid) (g-PGA), hyaluronic acid (HA), and oligonucleotides have been deposited onto the polycation/pDNA (or cationic lipid/pDNA) polyplexes to recharge their surface to negative, and to effectively diminish the adverse interactions. 8-10 HA is a natural linear polysaccharide, which has pivotal roles in various biological functions.11 Moreover, HA has many advantages such as biocompatibility, non-immunogenicity and highly efficient target-specific delivery.12 Due to its excellent biocompatibility and biodegradability, HA has been extensively investigated for biomedical applications such as tissue engineering, drug delivery, and molecular imaging.13, 14 Most important of all, HA has attracted much attention for site-specific drug delivery to tumor because of its high affinity towards CD44 receptor (the cell adhesion protein family), RHAMM (receptor for HA-mediated motility), and ICAM-1 (intercellular adhesion molecule-1), which are over-expressed in various tumor cells.15 Therefore, it is proposed that the specific binding of HA to its receptor CD44 could be utilized for therapeutic application targeting to CD44-positive cancer cells. CD44 is a multifunctional cell surface protein involved in proliferation and differentiation,


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angiogenesis and signaling. The expression of CD44 in several types of human tumors and particularly in cancer stem cells, represent an appealing target for drug delivery in the treatment of cancer.16 The effective targeting of malignant cell surface antigens is essential in cancer therapy. Thus, CD44 overexpressing cells were usually used as model cancer cells for research. The aim of this study was to develop HA/RHB/pDNA nanoassemblies based on electrostatic coating as intelligent gene carriers for gene delivery to CD44-positive cancer cells, which are capable of inducing highly efficient gene transfection with low cytotoxicity. Therefore, RHB with disulfide content, a promising biodegradable polycation, was synthesized with bisacrylamide and amine monomer by Michael addition copolymerization. HA/RHB/pDNA nanoassemblies were developed by coating the RHB/pDNA nanoassemblies with HA by electrostatic adsorption. HA/RHB/pDNA nanoassemblies were characterized by morphology, particle size and zeta potential, and stability in vitro. Biosafety of RHB/pDNA and HA/RHB/pDNA nanoassemblies was evaluated by cytotoxicity, erythrocytes agglutination, and hemolytic in vitro. Moreover, we also compared the transfection activity of HA/RHB/pDNA nanoassemblies in B16F10 (CD44+) and NIH3T3 (CD44-) cells. In addition, the cellular uptake and targeting, accumulation and biodistribution, and gene transfection of HA/RHB/pDNA nanoassemblies in vivo were also investigated with Balb/c mice model bearing CD44-positive pulmonary tumors. RESULTS AND DISCUSSION Synthesis and characterization of RHB. Poly (amido amine)s with bioreducible disulfide linkages were reported to be promising polycations for gene delivery with high transfection efficiency and low cytotoxicity in vitro.17, 18 In this study, RHB was synthesized by Michael addition copolymerization between trifunctional amine (AEPZ) monomer and two bisacrylamide (CBA and MBA) monomers



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according to the scheme in Figure 1. The crosslinking of CBA and MBA (CBA: MBA=1: 2) via triamine AEPZ occurred by the reaction of acrylamide groups with primary and secondary amines of AEPZ. The structure and molecular weight of RHB can be controlled by molar ratios of monomers. Reaction of a triamine monomer and bisacrylamide monomers at equal molar ratio will produce linear polymers, while hyperbranched polymers will be produced at the molar ratio of 1:2. Moreover, the reducible disulfide chain density of RHB can be further varied by the molar ratio of CBA to MBA. The hyperbranched structure of successful production RHB containing disulfide linkages was confirmed by 1

H NMR analysis (Figure 2). In the 1H NMR spectra, a characteristic peak of the acrylamide groups in

CBA and MBA were found at δ=5-6.5 ppm. However, the acrylamide peaks disappeared in the spectrum of final product RHB, indicating that the acrylamide groups had conjugated successfully with the amino groups of AEPZ. The molecular weight of the RHB was measured by gel permeation chromatography. The weight-average molecular weight (Mw) was 60 kDa. The polydispersity index (PDI) was 1.6, indicating a good control of the polymerization.

Figure 1. The synthetic schemes of RHB.


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Figure 2. The 1H NMR spectrums of CBA (A), MBA (B), and RHB (C). It is often believed that buffering capacity of cationic polymers is an important factor for endosomal escape and efficient intracellular delivery of polymer/pDNA polyplexes due to the “proton sponge effect”.19, 20 Evidences showed that the acidic pH in lysosome vesicles can result in protonation of the amine groups of cationic polymers, when the delivery vector enters the lysosome. That can lead to vesicle swelling, rupture and eventually polymer/pDNA polyplexes are released from the entrapment of endosome/lysosome vesicles.21, 22 Therefore, the buffering capacity of cationic polymers is related to the prevention of pDNA degradation in lysosomes and the ease of delivery of pDNA into nucleus from lysosomes which results in better transfection activity. In other words, the transfection potential of cationic polymers is found to be related to their buffering capacity. Here, the buffering capacity of RHB in 150 mM NaCl solution was determined by acid–base titration by adding 1 M HCl to the polymer solution and expressed as the percentage of amine groups being protonated in the pH range (from pH 7.4 to 5.1), which mimic the pH range from the high pH extracellular environment to the low pH endosomal environment. NaCl solution was used as a control. As shown in Figure S1, RHB has a lower slope compared with NaCl solution in the pH 7.4–5.1, indicating that RHB showed excellent buffering



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capacity within the pH range, which had displayed 47.1% protonation. NaCl containing no amino groups in the structures demonstrated no buffering effect in the pH range. A previous report suggested that the high buffering capacity will facilitate the endosomal escape of the nanoassemblies, leading to enhanced transfection efficiency. 23 Preparation and characterization of RHB/pDNA and HA/RHB/pDNA nanoassemblies. RHB/pDNA and HA/RHB/pDNA nanoassemblies were spontaneously formed by electrostatic interaction. TEM was used to examine the morphology of binary and ternary nanoassemblies. As shown in Figure S2, the representative TEM images of RHB/pDNA (at weight ratio of 5:1) (Figure S2A) and HA/RHB/pDNA (at weight ratio of 5:5:1) (Figure S2B) nanoassemblies exhibited spherical morphology with moderate and uniform particle size, and no significant differences in morphology were observed between binary and ternary nanoassemblies. Moreover, no obvious aggregation was visible in Figure S2. Two major determinants for polyplexes used as gene carriers to be internalized into cells are the size and zeta potential of the polymerized particles. The formation, particle size, and zeta potential of polyplexes depend strongly on the ratios of the composition. Therefore, weight ratios of 1:1~50:1 for RHB/pDNA nanoassemblies and 1:5:1~5:5:1 for HA/RHB/pDNA nanoassemblies were selected as representative compositions to characterize the effect of increasing weight ratio on the properties of the nanoassemblies. The particle size and zeta potential of self-assemble nanoassemblies at various weight ratios were measured by DLS technique (Table 1). The results showed that increasing the amount of RHB with respect to a fixed amount of pDNA resulted in an increase of the zeta potential from 12.1 ± 2.8 mV to 21.5 ± 2.1 mV, indicating that all of the negative charges of pDNA were neutralized by the positive charges of RHB. All of the nanoassemblies maintained nanometer size (100-500 nm) within


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the tested weight ratios range. The particle size of RHB/pDNA nanoassemblies decreased and then increased with an increase of weight ratios, and the smallest particle size (102.4 ± 10.3 nm) was observed at weight ratio of 5: 1 and the corresponding zeta potential was found to be 15.3 ± 1.4 mV, which were considered as optimum value for further analysis. Moreover, the addition of negatively charged HA has increased the size of the nanoassemblies up to 165.9 ± 8.1 nm ~ 187.6 ± 11.4 nm and decreased the zeta potential to -3.9 ± 0.7 mV ~ -9.1 ± 0.3 mV, suggesting effective formation of the ternary nanoassemblies. These results were consistent with other reports.7, 24 Table 1 Mean particle size and zeta potential of RHB/pDNA and HA/RHB/pDNA nanoassemblies at various weight ratios (n = 5). Nanoassemblies

RHB/pDNA

HA/RHB/pDNA

Weight ratios

Particle size (nm)

Zeta potential (mV)

1:1

500.7 ± 28.6

12.1 ± 2.8

2.5:1

216.6 ± 8.2

14.2 ± 1.3

5:1

102.4 ± 10.3

15.3 ± 1.4

10:1

118.5 ± 4.7

17.2 ± 0.8

25:1

132.4 ± 5.1

18.9 ± 1.2

50:1

156.7 ± 12.9

21.5 ± 2.1

1:5:1

165.9 ± 8.1

-3.9 ± 0.7

2.5:5:1

171.8 ± 7.1

-6.7 ± 0.6

5:5:1

187.6 ± 11.4

-9.1 ± 0.3

Reductive degradation of RHB and pDNA release study. RHB/pDNA nanoassemblies were prepared at weight ratios of 2.5:1, 5:1, 10:1, and 50:1. The sensitivity of RHB/pDNA nanoassemblies to reducing environment was simulated by incubation with the reducing agent DTT (2.5 mM) for 30 min. Agarose gel retardation assay was performed to confirm the disassembly of RHB/pDNA nanoassemblies induced by disulfide reduction in RHB (Figure S3). As shown in Figure S3A, RHB could condense pDNA effectively in absence of DTT and pDNA migration in the gel remained fully retarded. As the disulfide bonds in RHB may be cleaved into fragments in the reducing environment of DTT, the resultant short cationic residues then display lower affinity to pDNA, which are not capable



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of maintaining pDNA in the condensed state. When the nanoassemblies were incubated with DTT, mimicking the reducing intracellular environment, pDNA was completely released from the fragmented nanoassemblies. As shown in Figure S3B, the RHB/pDNA nanoassemblies showed pDNA release at all of the test weight ratios, indicating that RHB can be easily and reducibly biodegraded in the intracellular environment, which provide a favorable physical toxicity property for its application in

vivo. pDNA condensing ability of RHB and stability of nanoassemblies. The pDNA condensation capacity of polycations and the stability of polycation/pDNA polyplexes are prerequisite for successful gene delivery. Therefore, the formation and stability of RHB/pDNA and HA/RHB/pDNA nanoassemblies were confirmed by agarose gel retardation assay (Figure S4). The pDNA binding and condensation capacity of RHB was first determined at various RHB/pDNA weight ratios. As shown in Figure S4A, the interaction based electronic absorption between RHB and pDNA was not enough to against the applied electric field at weight ratio of 1:1, even though RHB could condense pDNA to form nanoassemblies with 500.7 ± 28.6 nm of particle size and positive zeta potential. The results demonstrated that the formed RHB/pDNA nanoassemblies at weight ratio of 1:1 were thought to be loose and physicochemically unstable. Moreover, there was no free pDNA band visible in lanes 4-8 compared to the lane 2 containing only pDNA, indicating that RHB could effectively bind and condense pDNA even which could inhibit pDNA electrophoresis behavior completely. Some researchers had shown that addition of polyanion usually led to the decomposition of the polycation/pDNA polyplexes by competitive dissociation of the pDNA molecule from the polycation.25 To investigate whether the addition of anionic HA will decompose RHB/pDNA nanoassemblies, the


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release of pDNA from HA/RHB/pDNA nanoassemblies in the presence of various ratios of HA was detected by gel retardation assay (Figure S4B). The complete retardation of RHB/pDNA nanoassemblies could be observed when the weight ratio of RHB/pDNA was 5:1 (lane 3). However, when the RHB/pDNA nanoassemblies were coated with HA, electrophoretic images (lane 4-6) showed that HA/RHB/pDNA nanoassemblies at various weight ratios were retarded in the loading wells completely and no pDNA band was observed in the presence of various amounts of HA in comparison to RHB/pDNA nanoassemblies. The results indicated that RHB could bind pDNA strongly and anionic shielding did not decompose the RHB/pDNA nanoassemblies within the experimental range. The dissociation of polymer/pDNA polyplexes must occur within the nucleus for a successful transfection.26 Otherwise, the released pDNA will be degraded by enzyme in vivo. In addition, negatively charged proteins in circulation system will compete the binding to polycation with pDNA, which results in rapid clearance by macrophages and reduction of delivery efficiency. The stability of RHB/pDNA and HA/RHB/pDNA nanoassemblies in vivo was simulated by treatment with DNase I, sodium heparin, and serum in vitro. Gel retardation assay was performed after the nanoassemblies were incubated with different concentrations of DNase I to determine the protection from enzymatic degradation. As shown in Figure S4C, both RHB/pDNA and HA/RHB/pDNA nanoassemblies could protect most of the pDNA from enzymatic degradation because there were not only pDNA bands but also visible smears resulting from pDNA degradation in the corresponding lanes. However, HA/RHB/pDNA nanoassemblies showed stronger enzymatic protection, shielding pDNA against DNase I activity than RHB/pDNA nanoassemblies, since fewer smears were visible throughout the corresponding lanes with the increasing of amounts of HA. Sodium heparin is often used to simulate negatively charged macromolecules in the blood for evaluating the anti-dissociation characteristics of



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polycation/pDNA polyplexes in vitro. As shown in Figure S4D, the dissociation effect of sodium heparin is concentration-dependent. No free pDNA bands could be observed when binary or ternary nanoassemblies were treated with sodium heparin at 10 and 50 mg/mL, indicating that sodium heparin couldn’t dissociate the RHB/pDNA and HA/RHB/pDNA nanoassemblies. However, sodium heparin did slightly change pDNA electrophoresis shift at 100 mg/mL, because there were pDNA bands visible resulting from dissociation of nanoassemblies. The possibility of destabilization of nanoassemblies by 10%, 20% and 40% FBS was showed in Figure S4E. Electrophoretic images showed that RHB/pDNA and HA/RHB/pDNA nanoassemblies at various weight ratios were retarded in the loading wells completely. The nanoassemblies incubated with increasing amounts of serum didn’t result in the release of pDNA. It seemed that serum didn’t affect pDNA complexation ability. The results indicated that RHB could condense pDNA completely and protect pDNA from being digested by serum in the blood. Cytotoxicity in vitro. Cationic polymers are considered to be able to improve gene transfection efficiency by the electrostatic interactions between polyplexes and cell membrane.27 But the strong interactions usually cause cell membrane damage to induce high cytotoxicity.20,

28, 29

Moreover,

erythrocytes aggregation and hemolysis may occur after systemic injection because of the interactions between polyplexes and blood cells. Therefore, the cytotoxicity, erythrocytes agglutination and hemolysis of RHB/pDNA and HA/RHB/pDNA nanoassemblies were evaluated in vitro (Figure 3). Cytotoxicity of RHB/pDNA and HA/RHB/pDNA nanoassemblies was evaluated by MTS assay in B16F10 cells. LipofectamineTM 2000 was used as control. As presented in Figure 3A, the cytotoxicity of RHB/pDNA nanoassemblies increased with the increasing of RHB/pDNA weight ratios, and RHB/pDNA nanoassemblies showed extremely high cytotoxicity (cell viability < 50%) when the


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RHB/pDNA weight ratios were higher than 25:1. The cytotoxicity of RHB/pDNA nanoassemblies at weight ratio of 10:1 or less was significantly lower than LipofectamineTM 2000. As mentioned above, RHB used in this study has biodegradable disulfide bonds, which could reduce the cytotoxicity of RHB in intracellular environment. However, it is expected that during the early stages of incubation, the plasma membrane disruption by high charge density of extracellular polycations is a significant contributing factor for the observed cytotoxicity. The presence of disulfide bonds in RHB is unlikely to reduce the cytotoxicity of RHB in extracellular environment. According to the reports, RHB with no disulfide content exhibited a higher cytotoxicity than RHB with disulfide bonds. As expected, HA/RHB/pDNA

nanoassemblies

showed

significant

lower

cytotoxicity

than

RHB/pDNA

nanoassemblies with the same quantity of RHB for pDNA complexation, regardless of the HA/RHB weight ratios and incubation time (Figure 3B). The results of cytotoxicity assay indicated that HA coating could significantly reduce cytotoxicity of RHB. It was reported that cationic polyplexes and lipoplexes often caused systemic toxicity and agglutination of negatively charged erythrocytes and albumin because of the strong electrostatic interactions.30, 31 These agglutinations often lead to rapid elimination and adverse effects, such as embolism and inflammatory reaction. Erythrocytes agglutination was evaluated by incubating binary or ternary nanoassemblies with erythrocytes. As shown in Figure 3C, erythrocytes incubated with RHB/pDNA nanoassemblies at weight ratio of 5:1 displayed obvious aggregation as compared to erythrocytes incubated with PBS. In contrast, HA/RHB/pDNA nanoassemblies (at weight ratio 1:5:1, 2.5:5:1, and 5:5:1) progressively decreased erythrocytes aggregation with the increasing amounts of HA, suggesting that the effect was concentration-dependent. When the weight ratio of HA/RHB/pDNA reached 5:5:1, any erythrocytes agglutination activity was not observed.



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Hemolytic property in vitro was used to investigate membrane disruption effect of nanoassemblies. Experiments included negative control (0.9% saline) and positive control (Triton X-100), and the degree of hemolysis caused by nanoassemblies exposed to dilute rabbit RBC for 4 h of incubation was shown in Figure 3D and E. The hemolysis activity of RHB/pDNA nanoassemblies increased as a function of increasing the weight ratios of RHB/pDNA (Figure 3D). In other words, increasing the mass concentration of RHB in the 0.9% saline/blood mixture with respect to a fixed amount of pDNA resulted in an increase of the percentage of hemolysis, which was a concentration-dependent manner. The hemolytic activity of binary nanoassemblies may be caused by interaction of RHB with the erythrocyte membrane. In general, HA/RHB/pDNA nanoassemblies exhibited significantly lower hemolysis than RHB/pDNA nanoassemblies at equivalent mass concentration of RHB (Figure 3E). This could be attributed to the HA shielding effect. Hemolysis caused by RHB/pDNA nanoassemblies progressively increased from approximately 1.78 ± 0.23% to 16.6 ± 6.50%. According to the criterion, percent hemolysis > 5% indicates that the test material causes damage to RBC. In comparison, a much lower hemolytic activity was measured for HA/RHB/pDNA nanoassemblies, while approximately 1.48 ± 1.02% hemolysis was observed at weight ratio of 1:5:1 and even down to less than 1% hemolysis at weight ratio of 2.5:5:1. The presence of HA on ternary nanoassemblies at weight ratio of 5:5:1 showed no significant difference in hemolysis compared with negative control. In conclusion, the strong positive charges of RHB/pDNA nanoassemblies were known to cause cytotoxicity by interaction with the negative surface of the cellular membrane. The addition of negatively charged HA effectively reduced the surface charges of the nanoassemblies from positive to negative, which must decrease the interaction of RHB with the blood components and reduce the cytotoxicity.


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Figure 3. The cytotoxicity, erythrocytes agglutination, and hemolysis evaluation of RHB/pDNA and HA/RHB/pDNA nanoassemblies in vitro. (A) Cytotoxicity of RHB/pDNA at weight ratios of 2.5:1, 5:1, 10:1, 25:1, and 50:1 against B16F10 cells; (B) Cytotoxicity of HA/RHB/pDNA nanoassemblies at weight ratios of 1:5:1, 2.5:5:1, and 5:5:1 against B16F10 cells; (C) Erythrocytes agglutination of RHB/pDNA and HA/RHB/pDNA nanoassemblies. Agglutination was observed by microscopy (200 × magnification). (a) PBS, (b) RHB/pDNA nanoassemblies at weight ratio of 5:1, (c-e) HA/RHB/pDNA nanoassemblies at weight ratios of 1:5:1, 2.5:5:1, and 5:5:1; (D) Hemolytic activity induced by



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RHB/pDNA nanoassemblies at weight ratios of 2.5:1, 5:1, 10:1, 25:1, and 50:1 in vitro; (E) Hemolytic activity induced by HA/RHB/pDNA nanoassemblies at weight ratios of 1:5:1, 2.5:5:1 and 5:5:1 in vitro. 0.9% saline and Triton X-100 were used as negative control and positive control, respectively. Each bar represents the mean ± SD (n = 5). *: p < 0.05, **: p < 0.01, ***: p < 0.001. Transfection efficiency of RHB/pDNA and HA/RHB/pDNA nanoassemblies in vitro. The transfection activity of RHB/pDNA nanoassemblies was investigated using pEGFP-N2 as reporter gene to identify a suitable RHB/pDNA weight ratio for further studies. The initial transfections were evaluated with fresh RHB/pDNA nanoassemblies prepared at weight ratios of 2.5:1, 5:1, 10:1, 25:1, and 50:1 in B16F10 cells and LipofectamineTM 2000/pDNA complexes were used as positive control. The transfection efficiency of RHB/pDNA nanoassemblies in vitro presented as fluorescence images and percentage of EGFP-positive cells were shown in Figure 4. Theoretically, RHB/pDNA nanoassemblies with higher weight ratio should show higher gene transfection efficiency, presumably due to the formation of more stable nanoassemblies and more efficient cellular internalization with the high cationic charge density. In this study, the highest transfection efficiency of RHB/pDNA nanoassemblies in B16F10 cells was observed at weight ratio of 10:1 which was lower than that of LipofectamineTM 2000/pDNA complexes. Otherwise, there were no significant differences in the transfection efficiency of RHB/pDNA nanoassemblies at weight ratio of 5:1 and 10:1 in B16F10 cells. However, the cytotoxicity (Figure 3) in vitro gradually improved with increasing the weight ratios of RHB/pDNA, especially at weight ratios 25:1 and 50:1. Importantly, RHB/pDNA nanoassemblies at weight ratio of 5:1 showed lower cytotoxicity than RHB/pDNA nanoassemblies at weight ratio of 10:1 and LipofectamineTM 2000/pDNA complexes against B16F10 cells. Therefore, the final RHB/pDNA weight ratio was fixed at 5:1 in further studies.


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Figure 4. Transfection efficiency of RHB/pDNA nanoassemblies at various weight ratios (2.5:1, 5:1, 10:1, 25:1, and 50:1) in B16F10 cells. LipofectamineTM 2000/pDNA complexes were used as a positive control. (A) Representative fluorescent images of B10F10 cells treated with RHB/pDNA nanoassemblies. Original magnification: 20×. (B) Transfection efficiency of

RHB/pDNA

nanoassemblies evaluated by flow cytometry. Each bar represents mean ± SD (n=5). **: p < 0.01. It was proposed that the specific binding of HA to its receptor CD44 could be utilized for therapeutic targeting to CD44-positive cancer cells.32-34 Therefore, the transfection activity of HA/RHB/pDNA nanoassemblies was evaluated in CD44-positive B16F10 cells and CD44-negative



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NIH3T3 cells by fluorescence microscopy and flow cytometry to confirm the HA mediated delivery (Figure 5). Lipofectamine 2000/pDNA complexes were used as the positive control. As shown in Figure 5A, stronger green fluorescent signals were observed in the CD44-positive B16F10 cells than CD44-negative NIH3T3 cells which were treated with different weight ratios of RHB/pDNA and HA/RHB/pDNA nanoassemblies. This may contribute to the different transfection properties of B16F10 and NIH3T3 cells. Fluorescence images demonstrated that RHB/pDNA nanoassemblies at weight ratio of 5:1 showed significantly lower GFP expression compared with LipofectamineTM 2000/pDNA complexes in both B16F10 and NIH3T3 cells. As expected, HA coating can significantly enhance the transfection efficiency of RHB/pDNA nanoassemblies in CD44-positive B16F10 cells, and the fluorescence intensity was increased along with the increasing of HA ratio in the test range. However, HA significantly decreased the transfection efficiency of RHB/pDNA nanoassemblies in CD44-negative NIH3T3 cells. Transfection efficiency of the nanoassemblies was quantified by flow cytometry to confirm the expression of GFP, which showed the same tendency with fluorescence images. As presented in Figure 5B, in B16F10 cells, the GFP expression of HA/RHB/pDNA nanoassemblies was significantly higher than that of RHB/pDNA nanoassemblies. For example, the transfection efficiency of HA/RHB/pDNA nanoassemblies at weight ratio of 5:5:1 in B16F10 cells was 78.9 ± 3.81%, which was approximately 1.43-fold higher than that of RHB/pDNA nanoassemblies 55.3 ± 4.04% (p < 0.001). In contrast, in NIH3T3 cells, the transfection efficiency of RHB/pDNA nanoassemblies at weight ratio of 5:1 was 18.5 ± 3.23%, while that of HA/RHB/pDNA nanoassemblies at weight ratio of 5:5:1 was only 3.87 ± 1.25%, which decreased by 4.8-fold (p < 0.001). To further confirm the specific interactions of HA with CD44 receptors, competitive inhibition assay was performed in B10F10 and NIH3T3 cells with pre-treatment of excess anti-CD44 antibody.


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As shown in Figure S5, pre-treatment of excess anti-CD44 antibody significantly reduced the transfection efficiency of HA/RHB/pDNA nanoassemblies in B16F10 cells. The quantitative inhibitory results showed that the transfection efficiency of HA/RHB/pDNA nanoparticles reduced to 44.2 ± 9.91% of the original levels in B16F10 cells. As expected, there was no difference observed in the transfection efficiency of CD44 negative NIH3T3 cells after treatment of anti-CD44 antibody. Efficient transfection of HA-based gene delivery systems in CD44-overexpressing cells has been demonstrated in many studies.11, 12, 33 Obviously, the significantly different transfection efficiency of HA/RHB/pDNA nanoassemblies between B16F10 and NIH3T3 cells and competitive inhibition assay indicated that HA played an important role in gene transfection. These results confirmed that the high transfection efficiency of HA/RHB/pDNA nanoassemblies in B16F10 cells was implemented due to the enhanced internalization of the nanoassemblies through CD44 receptor-mediated endocytosis. Therefore, the HA/RHB/pDNA nanoassemblies at weight ratio of 5:5:1 with appropriate transfection efficacy and cytotoxicity will be used for gene delivery in vivo.



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Figure 5. Transfection efficiency of HA/RHB/pDNA nanoassemblies at various weight ratios (0:5:1, 1:5:1, 2.5:5:1, and 5:5:1) in B16F10 and NIH3T3 cells. LipofectamineTM 2000/pDNA complexes were used as a positive control. (A) Representative fluorescent images of B16F10 and NIH3T3 cells treated with HA/RHB/pDNA nanoassemblies. Original magnification: 20×. (B) Transfection efficiency of HA/RHB/pDNA nanoassemblies in B16F10 and NIH3T3 cells quantified by flow cytometry. Each bar represents the mean ± SD (n = 5). *: p < 0.05, ***: p < 0.001. Serum-resistant transfection in vitro. It has been reported that transfection activity of polycations is sensitive to the presence of serum. Inefficient gene delivery by cationic polymeric carriers in serum-containing environment has largely limited their further gene therapy application in vivo. In this study, the effect of serum on the transfection efficiency of RHB/pDNA and HA/RHB/pDNA nanoassemblies was investigated in B16F10 cells in the presence of different concentrations of serum (0%, 10%, 20%, and 40% FBS) (Figure 6). In agreement with studies by other groups on polycation/pDNA,35,

36

in the presence of serum, the transfection efficiency of RHB/pDNA

nanoassemblies and LipofectamineTM 2000/pDNA complexes drastically suppressed even with 10% (v/v) of FBS. In contrast, fluorescence images of B16F10 cells which transfected with HA/RHB/pDNA


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nanoassemblies showed significant higher fluorescence intensity in comparison with RHB/pDNA nanoassemblies in the presence of serum, suggesting that HA/RHB/pDNA nanoassemblies expressed much more GFP than RHB/pDNA nanoassemblies, and also indicating that serum attenuated slightly the transfection efficiency of the HA/RHB/pDNA nanoassemblies. The results analyzed from the flow cytometry in Figure 6B were in accordance with fluorescent images results in Figure 6A. The binary and ternary nanoassemblies showed the highest transfection efficiency (56.8 ± 4.58) % and (78.3 ± 3.54) % in B16F10 cells with serum-free media, respectively. The HA/RHB/pDNA nanoassemblies still maintained the high transfection efficiency (59.3 ± 2.25%) even in the presence of up to 40% serum, in contrast to the decreased transfection efficiency (14.7 ± 3.51%) by RHB/pDNA nanoassemblies under the same conditions. The inhibition effect of serum in gene transfection has been considered to be one of the limitations for the application of cationic non-viral gene delivery systems in vivo.10, 37, 38 It had been explained that negatively charged biomacromolecules in systemic circulation such as serum could destroy the stability of polycation/pDNA complexes because of the non-specific interaction between polycation and serum. Binding of serum to cationic polyplexes may result in particle decomplexation or aggregation, which in turn contributes to inefficient gene transfection. These results indicated that polyanionic shielding maintained high transfection efficiency of the cationic nanoassemblies in the presence of serum. HA/RHB/pDNA ternary nanoassemblies may have predominance as a gene carrier for application in vivo as compared with RHB/pDNA nanoassemblies.



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Figure 6. Serum-resistant transfection of HA/RHB/pDNA nanoassemblies compared with RHB/pDNA nanoassemblies in vitro. (A) Representative fluorescent images of B16F10 cells treated with (a) RHB/pDNA (w/w, 5:1) nanoassemblies, (b) HA/RHB/pDNA (w/w/w, 5:5:1) nanoassemblies, and (c) LipofectamineTM 2000/pDNA complexes in the presence of 0%, 10%, 20%, and 40% FBS, respectively. Original magnification: 20 ×. (B) The percentage of B16F10 cells expressing GFP determined by flow cytometry. The data were presented as mean ± SD (n = 5). Intracellular distribution and co-localization. It is well known that endosome/lysosome system is the major challenge for efficient gene delivery. Endosomal/lysosomal escape based on the “proton sponge effect” is crucial to enhance the efficacy of gene delivery systems.1, 35 To investigate the intracellular


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distribution and localization of HA/RHB/pDNA nanoassemblies after cellular uptake, confocal laser scanning microscopy was used to collect images of the cells incubated with nanoassemblies for different time durations. As shown in Figure 7, large amount of green fluorescence could be observed at all the time points tested in this study. The nanoassemblies interacted with the cellular membrane at 2 h after nanoassemblies addition as majority of the green fluorescence was found to be localized at the cell surface. Moreover, a significant fraction of nanoassemblies was confined within lysosomal compartments as evidenced colocalization analysis at 2 h. Perfect colocalization of pDNA (green) and late endosomes/lysosomes (red) produced yellow pixels due to the overlapping of the green and red fluorescence. Nanoassemblies progressively arrived at the late endosomes/lysosomes as more yellow pixels were observed at 12 h after incubation started. After 12 h, fraction of the nanoassemblies successfully escaped from the acidic vesicles as green fluorescence was detected in the cytoplasm and around the nucleus. Yellow pixels could still be seen at this time point which represents partial escape of nanoassemblies from late endosomes/lysosomes. It obviously showed that the incubation of cells with nanoassemblies for 24 h resulted in separate localization of green and red fluorescence, and more green fluorescence co-localized in the nucleus. Therefore, these observations could suggest that the cellular uptake and endosomal escape of HA/RHB/pDNA nanoassemblies were time-dependent progresses. The carriers efficiently escaped from these degradative compartments, which is important for posterior transgene expression.



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Figure. 7. Intracellular distribution of HA/RHB/pDNA nanoassemblies in B16F10 cells. B16F10 cells were incubated with HA/RHB/YOYO-1-pDNA (green) nanoassemblies. At different time intervals after incubation, cells were stained with Lyso-Tracker Red (red, endosome-lysosome system) and Hoechst33342 (blue, nucleus) and imaged by confocal microscope. Yellow dots in merged images indicated YOYO-1-pDNA (green) trapped within acidic endosomes/lysosomes components (red). Effect of inhibitors on endocytosis and intracellular transport of HA/RHB/pDNA nanoassemblies. For successful gene delivery, the vectors must achieve internalization and intracellular transport towards the nucleus before gene expression. Elucidation of uptake mechanisms and intracellular fate of non-viral gene vectors is important for the design and optimization of the novel multifunctional delivery systems. Inhibitors against endocytosis and intracellular transport can be used to block the specific endocytic and transport pathway to confirm whether they are employed by the gene vectors. The internalization and transfection efficiency of HA/RHB/pDNA nanoassemblies by B16F10 cells in the presence of various inhibitors were determined by flow cytometry and the results were showed in Figure 6S. The counterparts in the absence of inhibitors were used as controls. As suggested by the results of preliminary test, the inhibitory function of various inhibitors was concentration-dependent and the inhibitors didn’t show any significant cytotoxicity in the test concentration ranges. A variety of forms of endocytosis have been demonstrated to be involved in the cellular uptake of


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polyplexes.39, 40 In order to evaluate the relative contribution of the potential endocytic pathways in HA/RHB/pDNA nanoassemblies-mediated uptake and transfection, the cells were treated with different inhibitors against clathrin-mediated endocytosis (CME), caveolae-mediated endocytosis (CvME), and macropinocytosis (MP).35 The effective inhibition of CME was tested by using the cationic amphiphilic drug CPZ and hypertonic glucose, while filipin III and genestein were used to discern the role that CvME may play in cellular uptake. DMA (an inhibitor of sodium-proton exchange) and CyD (an F-actin depolymerizing agent) were used to inhibit the macropinocytosis. As shown in Figure 6SA, all inhibitors could significantly decrease the cellular uptake of HA/RHB/pDNA nanoassemblies. Relative to the control, hypertonic glucose showed the strongest inhibitory effect, the uptake of nanoassemblies was reduced by 55.9 ± 4.15%. The cellular uptake of nanoassemblies decreased relatively to 56.4 ± 3.92% in the presence of CPZ. Uptake of the nanoassemblies in cells treated with filipin III and genistein also decreased by 53.1 ± 6.41% and 44.5 ± 8.86%, respectively. Cells pre-treated with DMA and CyD also resulted in significant inhibition of the internalization of nanoassemblies, the reduction was 41.1 ± 3.07% and 53.4 ± 3.91%, respectively. These results clearly suggested that the internalization of nanoassemblies took place by a combination of mechanisms. The CME and CvME were involved in the internalization of nanoassemblies and MP also played an important role in the endocytosis of nanoassemblies. It is unknown whether the nanoassemblies can transfect effectively after entering cells through endocytosis, as pDNA is likely to be degraded by intracellular enzymes before successful expression. Therefore, the effects of endocytosis inhibitors on transfection efficiency were also investigated (Figure 6SA). Inhibition of CME by CPZ decreased the transfection efficiency of the nanoassemblies to 52.3 ± 2.73%. Transfection efficiency of cells pre-treated by hypertonic glucose was decreased by 30.2 ±



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5.69%. CvME inhibitors filipin III and genistein also had significant inhibitory effects on the transfection efficiency, and the degree of inhibition was 49.1 ± 9.72% and 31.7 ± 3.76%, respectively. The transfection efficiency of nanoassemblies was significantly inhibited to 50.6 ± 4.86% in the presence of CyD. DMA also had different degree of inhibitory, while the transfection efficiency was suppressed by 30.7 ± 3.76%. These results indicated that endocytosis of the nanoassemblies composed of three mechanisms: CME, CvME and MP, which played a major role in the subsequent transgene expression. After entering cells by endocytosis, the cellular process (including endosomal escape, transport, and nuclear localization) is one of the most important steps for non-viral gene delivery. All of these challenges are considered to be particularly limiting to transfection efficiency. Especially, intracellular endosomal escape would be expected to be the major factor controlling the fate of the introduced gene and the efficiency of expression.38, 41 The endosome/lysosome system acidification was inhibited using NH4Cl and monensin to examine its function in the effective transfection of the nanoassemblies. As shown in Fig. 6SB, both NH4Cl and monensin dramatically decreased the transfection efficiency of the nanoassemblies to 59.7 ± 7.05% and 26.5 ± 7.35%, respectively. NCZ, a microtubule depolymerizing agent, was used to evaluate the contribution of microtubules in the transfection efficiency of the nanoassemblies. NCZ significantly decreased the transfection efficiency to 45.6 ± 2.18%. SOV, which primarily inhibits cytoplasmic dynein ATPase activity, significantly decreased the transfection efficiency of the nanoassemblies to 51.0 ± 6.51%. In contrast, there was no inhibitory effect on the transfection efficiency of the nanoassemblies observed for the cells pre-treated with monastrol, which inhibits the kinesin Eg5 and roscovitine (ROS) to inhibit the kinase CDC2A. Thus, these results suggested that microtubule and cytoplasmic dynein played vital roles in controlling the intracellular


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translocation of exogenous pDNA for the effective transfection, but kinesin wasn’t involved in the process of internalization of nanoassemblies. The cellular uptake and transport mechanisms of non-viral vectors are the most essential to their transfection efficiency. Unfortunately, there are some disadvantages for the commonly used inhibitor tools.42 For example, previous researches showed that the inhibitors of the specific endocytic pathway always influence on other pathways, and the inhibitors can block different endocytic mechanisms in different cell types.43 It is necessary to study further for a better understanding, and the findings may contribute to the emerging of the HA/RHB/pDNA nanoassemblies. CD44 targeting and cellular uptake of HA/RHB/pDNA nanoassemblies in vivo. In order to evaluate cellular uptake and CD44 receptor specific targeting of HA/RHB/pDNA nanoassemblies in vivo, C57BL/6 mice bearing pulmonary tumors were administrated with YOYO-1-labeled naked pDNA, RHB/YOYO-1-labeled pDNA or HA/RHB/YOYO-1-labeled pDNA nanoassemblies by intravenous injection on 10th day after B16F10 cells inoculation. PBS was used as a negative control. Mice were sacrificed at different time point after intravenous administration and frozen slices of lung tissues were obtained to observe in vivo lung distribution and cellular uptake characteristics of YOYO-1-labeled nanoassemblies under fluorescence microscopy. Fluorescent images of frozen slices at 30 min, 4 h, and 24 h after injection of PBS, naked pDNA solution, RHB/pDNA or HA/RHB/pDNA nanoassemblies were shown in Figure 8. Due to the poor stability of nucleic acid in blood, the fluorescence signal was almost undetectable in the YOYO-1-labeled naked pDNA group at all of the time points. It was reported that there are various kinds of nuclease existed in the blood, resulting in a rapid elimination of naked pDNA from the circulation. There were only a few green fluorescence signals distributed in lung tissues of RHB/YOYO-1-labeled pDNA nanoassemblies group. The fluorescence signals were



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relatively stronger than YOYO-1-labeled naked pDNA group and showed a time-dependent tendency. At 30 min post-injection, maximum fluorescent signals were exhibited and the signals decreased over time, suggesting that RHB/pDNA nanoassemblies could be internalized by cells to some degree, which mainly contributed to the accumulation in tumor region by EPR effect. But these binary nanoassemblies were still quite unstable in the circulation because of the large quantity of positive charges on the surface of the particles. HA/RHB/YOYO-1-labeled pDNA nanoassemblies group had strongest fluorescent signals at all survey points in the three treatment groups. Fluorescence signals of ternary nanoassemblies group reached a high level at 4 h post-injection and maintained at this level until 24 h. The higher cellular uptake of HA/RHB/pDNA nanoassemblies in vivo should be attributed to two reasons: (1) HA coating significantly reduced the surface charges of HA/RHB/pDNA nanoassemblies, leading to a higher stability in serum and longer circulation time as well as larger amount of accumulation in tumor tissue, which was beneficial for cellular internalization. (2) The interaction of HA with CD44 receptor promoted the cellular uptake in vivo.

Figure 8. CD44 targeting and cellular uptake of HA/RHB/pDNA nanoassemblies in vivo. C57BL/6 mice were administrated with PBS, naked pDNA, RHB/pDNA nanoassemblies and HA/RHB/pDNA nanoassemblies by intravenous injection, respectively. Lung tissues of C57BL/6 mice were collected at 30 min, 4 h and 24 h post-injection. Frozen slices of lung tissues were sectioned and the fluorescence


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distribution and cellular uptake of YOYO-1-labeled nanoassemblies in lungs were observed and imaged under fluorescence microscopy. Original magnification: × 20. Gene transfection and

distribution in vivo. To evaluate the transfection activity of

HA/RHB/pGL3-promoter nanoassemblies in vivo, the distribution of luciferase gene expression was analyzed following single injection of naked pGL3-promoter, RHB/pGL3-promoter nanoassemblies, and HA/RHB/pGL3-promoter nanoassemblies into C57BL/6 mice via tail vein. It was found that the RHB/pDNA nanoassemblies at weight ratio of 5:1 caused occasional mortality in both healthy mice group and tumor bearing mice group after injection. However, it should be noted that the death didn’t occur in the groups which were treated with ternary nanoassemblies at weight ratio of 5:5:1, indicating that HA coating could significantly reduce the toxicity of RHB/pDNA nanoassemblies in vivo. This phenomenon was consistent with the results of cytotoxicity, erythrocytes aggregation, and hemolysis in vitro. As shown in Figure 9, luciferase expression in vivo was determined to characterize the tissues distribution of nanoassemblies administered by intravenous injection. As expected, there was little luciferase expression to be found in naked pDNA treated healthy mice group and tumor bearing mice group, which indicated that naked pDNA was rapidly eliminated from the circulation. In contrast, it was found that luciferase expression was detectable in all organs including the heart, liver, spleen, lung, and kidney in the RHB/pDNA or HA/RHB/pDNA nanoassemblies treated mice groups. Theoretically, the organs distribution of non-targeting nanoparticles mainly depended on their particle size and surface charge.44 The RHB/pDNA nanoassemblies showed the maximum luciferase expression in lungs in both the healthy mice group and tumor-bearing mice group since they were unstable so that they formed aggregates in the blood. Most of the RHB/pDNA nanoassemblies were entrapped in the



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pulmonary capillaries of lung. Erythrocyte aggregation and subsequent trapping of the aggregates in the lung vasculature was probably responsible for the high lung disposition. High lung expression after intravenous administration has been reported for the majority of positively charged polyplexes.45 However, Luciferase expression of HA/RHB/pDNA nanoassemblies was more appeared in liver and spleen than lung in the healthy mice group, because the small (200 nm) and stable (negative surface charge) ternary nanoassemblies could escape from the lung capillaries and circulate through the whole body to the liver and spleen. Moreover, HA had significantly increased luciferase expression of RHB/pDNA nanoassemblies in various tissues as compared to the RHB/pDNA binary nanoassemblies without HA coating, which attributed to the stability (the appropriate particle size and negative charge) of HA/RHB/pDNA nanoassemblies in vivo. In addition, the luciferase activity in all organs of tumor-bearing mice group was higher compared to the healthy mice group, when they obtain the same treatment. This was probably due to the decreased immunity and scavenging ability of mononuclear phagocytic system in tumor-bearing mice. The difference of luciferase expression for RHB/pDNA and HA/RHB/pDNA nanoassemblies in tumor was remarkable, which was consistent with their different transfection capabilities in vitro. Luciferase expression of HA/RHB/pDNA nanoassemblies in tumor was 2.5-fold higher than that of RHB/pDNA nanoassemblies, indicating that the interaction of HA with CD44 receptor dramatically improved the gene-targeting delivery and gene expression in CD44-positive tumor in vivo. Therefore, HA/RHB/pDNA nanoassemblies showed the most potent transfection activity in vivo.


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Figure 9. The quantitative evaluation of luciferase expression in vivo. The PBS, naked pDNA, RHB/pDNA nanoassemblies, and HA/RHB/pDNA nanoassemblies were injected intravenously into healthy C57BL/6 mice and pulmonary tumor-bearing C57BL/6 mice, respectively. At 48 h after administration, mice were sacrificed and the main organs were dissected to quantify luciferase activity. The luciferase expression is plotted as light units per mg protein (RLU/mg protein). Each bar represents mean ± SD (n = 6). ***: p < 0.001. CONCLUSION In this study, RHB as a reducible hyperbranched polycation was synthesized for developing CD44-positive tumor-targeted HA/RHB/pDNA nanoassemblies based on electrostatically coating as a promising and efficient non-viral gene delivery system with excellent biocompatibility in vitro and in vivo. The resultant particles were spherical in shape and uniform in size. The addition of HA markedly decreased the toxicity of RHB/pDNA nanoassemblies since negative charges on the surface, while performing higher transfection efficiency in B16F10 compared to LipofectamineTM 2000. Incorporation of HA in the RHB/pDNA nanoassemblies resulted in enhanced cellular uptake and stability in vivo. HA in the shell of the ternary nanoassemblies make the nanoassemblies deliver pDNA to the tumor site by passive and active targeting. Gene transfection in vivo indicated that the HA/RHB/pDNA



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nanoassemblies presented lower toxicity and higher luciferase expression in B16F10 tumor than binary nanoassemblies resulting from hydrophilic and negatively charged surface, longer circulation in blood, and effective accumulation at the tumor site by CD44 receptor targeting. Thus, HA/RHB/pDNA nanoassemblies may be useful as a gene delivery system for the delivery of therapeutic genes to treat CD44-overexpressing tumors in vivo. MATERIALS AND METHODS Materials.

Hyaluronic

N′-cystaminebisacrylamide piperazine

(AEPZ),

acid

(HA,

(CBA),

N,

MW=7.5

kDa,

Lifecore

N′-methylenebisacrylamide

4-Amino-1-butanol

(ABOL),

3-(4,

Biomedical (MBA),

Co.),

N,

1-(2-aminoethyl)

5-dimethyl-2-thiazolyl)-2,

5-diphenyltetrazoplium bromide (MTT), Chlorpromazine hydrochloride (CPZ), Filipin III, Genistein, 5-(N, N-Dimethyl) amiloride hydrochloride (DMA), Cytochalasin D (CyD), Nocodazole (NCZ), Monensin, sodium orthovanadate (SOV), and Monastrol were purchased from Sigma-Aldrich (St. Louis, MO, USA). YOYO-1, LysoTracker Red DND26, Hoechst33342, and LipofectamineTM 2000 transfection kit were obtained from Life Technologies (Burlington, ON, USA). RPMI medium 1640, fetal bovine serum (FBS), Trypsine-EDTA (0.25%), penicillin-streptomycin, and agarose were purchased from Gibco (BRL, MD, USA). Murine melanoma cells (B16F10) and murine embryonic fibroblast cells (NIH3T3) were obtained from Chinese Academy of Sciences Cells Bank (Shanghai, China) and routinely maintained in RPMI medium 1640 supplemented with 10% (v/v) FBS, 100 U/mL penicillin and 100 µg/mL streptomycin at 37 °C in a humidified 5% CO2 incubator. All experiments were performed on cells in the logarithmic phase of growth. C57BL/6 mice (Male, 5 weeks old and 18-22 g) were purchased from the Experimental Animal


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Centre of Fudan University (Shanghai, China), and maintained under standard housing conditions. All animal experiments were performed in strict accordance with guidelines evaluated and approved by the ethics committee of Fudan University. All animal surgery was performed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering. Synthesis and characterization of RHB. RHB was synthesized by Michael addition copolymerization of triamine AEPZ and bisacrylamide monomers (CBA and MBA) according to the method previously described by D. Oupicky’s group.6, 23 The representation of RHB synthesis is shown in Figure 1. Briefly, CBA (0.260 g, 1.0 mmol) and MBA (0.308 g, 2.0 mmol) were added into a small vial containing AEPZ (0.193 g, 1.5 mmol) in methanol/water mixture (3.5 mL, 7/3 v/v). The polymerization reaction was carried out at 50 °C for 5 days, when the reaction mixture became highly viscous. Subsequently, additional ABOL was added to the reaction mixture to consume any residual acrylamide groups, and stirring was continued at 50 °C for 12 h. The RHB was obtained by freeze drying after extensive dialysis against distilled water acidified with HCl to pH 3 for 1 day and a final dialysis against distilled water for 2 days to remove low molecular weight fractions and to convert the RHB to its hydrochloride salts. RHB was characterized by 1H nuclear magnetic resonance (1H NMR) (400 MHz). The weight-average molecular weight (Mw) and polydispersity index (PDI) of RHB were determined by gel permeation chromatography (Agilent 1100 GPC, USA). Preparation and characterization of RHB/pDNA and HA/RHB/pDNA nanoassemblies. RHB and pDNA were dissolved in sodium acetate buffer with the final concentration of 1 µg/µL, respectively. For each preparation of RHB/pDNA nanoassemblies, appropriate amount of RHB solution was mixed with equal volume of diluted pDNA solution at weight ratios of 1:1, 2.5:1, 5:1, 10:1, 20:1, and 50:1, then vortexed slightly for 5 s, and incubated at room temperature for 10 min to ensure nanoassemblies



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formation. As a positive control, LipofectamineTM 2000/pDNA complexes were prepared according to the protocol provided by the manufacturer (Invitrogen). To prepare HA/RHB/pDNA nanoassemblies, HA solution was added to RHB/pDNA nanoassemblies at weight ratios of 1:1, 2.5:1 or 5:1 (HA/pDNA), mixed well and incubated for another 30 min at room temperature. The particle size and zeta potential of RHB/pDNA and HA/RHB/pDNA nanoassemblies were measured with the dynamic light scattering technique (DLS) using a Zetasizer Nano ZS (Malvern Instruments, Ltd., U K). Cell viability assay. The cytotoxicity of RHB/pDNA and HA/RHB/pDNA nanoassemblies in vitro was determined by MTT assay against B16F10 cells. B16F10 cells were seeded in 96-well plates at a density of 5×103 cells per well with complete medium and allowed to attach overnight. Then the cells were treated with serum-free medium (100 µL/well) containing RHB/pDNA or HA/RHB/pDNA nanoassemblies (0.2 µg pDNA/well) at various weight ratios for 4 h. Commercial LipofectamineTM 2000 was used as control in this study. The medium containing nanoassemblies or LipofectamineTM 2000/pDNA complexes was removed after 4 h and replaced with 100 µL of fresh complete medium, followed by an additional 48 h of culture at 37 °C. Then, 20 µL of MTT solution (5 mg/mL in PBS) was added to each well. After 4 h post-incubation, the medium including MTT was removed by aspiration and the formazan crystals formed were dissolved in 150 µL DMSO per well. The absorbance of each sample was then measured at a wavelength of 570 nm by a microplate reader (Synergy TM2, BIO-TEK Instruments Inc. USA). Cell survival percentages were calculated as the absorbance ratios of samples compared with the untreated control. The results were shown as a percentage of untreated cells with 100% viability. The relative cell viability (%) compared to untreated cells was calculated by Asample/Acontrol ×100. Erythrocyte agglutination and hemolysis evaluation in vitro. Erythrocytes were pelleted by


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centrifugation from fresh rabbit blood and washed 4-5 times in 0.9% saline containing anticoagulant heparin. To evaluate erythrocytes agglutination induced by RHB/pDNA or HA/RHB/pDNA nanoassemblies, 100 µL/well of 1% (v/v, diluted in 0.9% saline) erythrocytes suspension was added in 96-well plates and mixed with RHB/pDNA (at weight ratio of 5:1) and HA/RHB/pDNA (at weight ratios of 1:5:1, 2.5:5:1, and 5:5:1) nanoassemblies (0.2 µg pDNA/well) at a volume ratio (nanoassemblies/erythrocyte solution) of 1:1. After incubation for 15 min at room temperature, erythrocyte agglutination was observed and imaged by optical microscopy. Hemolysis assay was performed in vitro to evaluate the potential red blood cell (RBC) damage. Briefly, 100 µL of RHB/pDNA (at weight ratios of 2.5:1, 5:1, 10:1, 25:1, and 50:1) or HA/RHB/pDNA (at weight ratios of 1:5:1, 2.5:5:1, and 5:5:1) nanoassemblies (containing 0.2 µg pDNA) suspension were mixed with 100 µL of heparinized 1% (v/v, diluted in 0.9% saline) RBC at 37°C for 4 h with gentle inversion of the sample tubes every 30 min. Following the incubation, the samples were centrifuged at 2000 rpm for 5 min at room temperature. The absorbance (A) of the supernatant was analyzed at 545 nm to determine hemolysis response. RBC treated with 0.9% saline was used as a negative control and RBC lysed with 1% Triton X-100 was used as a positive control. All samples and positive/negative controls were analyzed in triplicate. Hemolysis (%) = [(ASample-ANegative) / (APositiveANegative)] ×100. Evaluation of transfection in vitro. B16F10 or NIH3T3 cells were seeded into 24-well plates at a density of 5×104 cells per well in 500 µL of complete medium, and allowed to adhere at 37 °C overnight (approximately 70-80% confluency). The pDNA encoding enhanced green fluorescent protein (pEGFP-N2, 4.7 kb) was used as a reporter gene. For transfection, RHB/pDNA (at weight ratios of 2.5:1, 5:1, 10:1, and 25:1, and 50:1) and HA/RHB/pDNA (at weight ratios of 1:5:1, 2.5:5:1,



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and 5:5:1) nanoassemblies containing 1 µg pEGFP-N2 were added to each well and incubated in the serum-free medium for 4 h. LipofectamineTM 2000/pDNA complexes were used as a positive control. Then the transfection medium was removed and replaced with 500 µL of fresh complete medium, and cells were incubated for another 48 h. The expression of green fluorescent protein (GFP) in the cells was observed under the fluorescence microscope (Leica, DMI 4000B, Germany). Transfection efficiency (the percentage of GFP-positive cells) was evaluated by flow cytometry (FACS Calibur, BD, USA). Serum resistance of RHB/pDNA and HA/RHB/pDNA nanoassemblies was studied in B16F10 cells which were exposed to the nanoassemblies for 4 h in culture medium containing various concentrations of FBS (10%, 20%, and 40%). Afterwards, the transfection medium was replaced with fresh culture medium. After incubation for 48 h, the transfected B16F10 cells expressing GFP were visualized and photographed by fluorescence microscopy (Leica, DMI 4000B, Germany). The transfection efficiency of the same corresponding cell population was detected by flow cytometry (FACS Calibur, BD, USA). Intracellular distribution and co-localization of the nanoassemblies. Intracellular distribution of nanoassemblies was evaluated by co-localization with confocal microscopy. B16F10 cells were seeded onto glass cover slips at 4 × 105 cells/well in 2 mL of medium in 6-well plate and cultured for 24 h. The culture medium was replaced by fresh serum free-medium containing HA/RHB/pDNA nanoassemblies (4 µg YOYO-1labeled pDNA/well). After treatment, the medium containing nanoassemblies was removed and the B16F10 cells were washed with cold PBS thrice at determined time interval. The cells were then sequentially incubated with DAPI (10 µg/mL) for 15 min and Lyso-tracker Red (50 nM) for 30 min to stain the cell nuclei and lysosomes, respectively. Thereafter, the cells were fixed with 4% (w/v) paraformaldehyde for 20 min and mounted in anti-fluorescence quenching mounting medium.


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Then the cells were observed and imaged with confocal laser scanning microscopy (Leica TCS SP5, Germany). Gene delivery and distribution in vivo. The pulmonary metastatic melanoma models were established by injecting 250 µL of B16F10 cells suspension (3.0 × 106 cells suspended in 1 mL PBS) intravenously through the tail vein of C57BL/6 mice. Gene delivery and distribution studies were performed after one week implanting. The accumulation and targeting effect of RHB/pDNA and HA/RHB/pDNA nanoassemblies in vivo were assessed by tumor-bearing C57BL/6 mice. 300 µL aliquot of freshly prepared naked YOYO-1-pDNA, RHB/YOYO-1-pDNA (at weight ratio of 5:1), or HA/RHB/YOYO-1-pDNA (at weight ratio of 5:5:1) nanoassemblies were injected intravenously via the tail vein of the C57BL/6 mice bearing pulmonary tumors at a dose of 100 µg pDNA per mouse with PBS serving as negative control. At different time points (30 min, 4 h, and 24 h) post-injection, the mice were sacrificed and the heart perfusion was performed with 0.9% saline and 4% paraformaldehyde. The tumor-bearing lungs were collected and fixed in 4% paraformaldehyde for 24 h, then sequentially dehydrated with 15% sucrose solution and 30% sucrose solution until subsidence. Afterward, the lungs were frozen in OCT (Sakura, Torrance, CA, USA) at -80 °C and cryosectioned into slides at a thickness of 10 µm. The sections were mounted in fluorescent mounting medium and imaged under a fluorescent microscope (Leica, DMI 4000B, Germany) to reveal the fluorescence distribution of nanoassemblies in lungs. Gene transfection and distribution in vivo were further performed by normal mice and tumor-bearing mice. The pDNA encoding for luciferase (pGL3-promoter, 5.01 kb) was used as a reporter gene. Mice were randomly assigned to the following treatment groups (n = 6): naked pGL3-promoter, RHB/pGL3-promoter and HA/RHB/pGL3-promoter nanoassemblies. 300 µL aliquot



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of freshly prepared RHB/pGL3-promoter (at weight ratio of 5:1) or HA/RHB/pGL3-promoter (at weight ratio of 5:5:1) nanoassemblies solution were immediately injected intravenously into the tail vein of C57BL/6 mice at a dose of 100 µg pDNA per mouse with naked pGL3-promoter serving as a control. For quantification of luciferase expression, the mice were sacrificed and the heart, liver, spleen, lung, and kidney were isolated at 48 h after the injection. The tissues were homogenized with 1 mL of lysis buffer (Promega, Madison, WI, USA). Homogenates were centrifuged at 15,000 rpm for 10 min at 4 °C and the supernatants were quantified for luciferase activity using a Luciferase Assay System. The tissue protein content was detected with BCA Protein Assay Kit (Beyotime Institute of Biotechnology, China). Results about luciferase activity were expressed as fluorescence intensity associated with 1 mg protein (RLU/mg protein). Statistical analysis. All results are reported as the mean ± standard deviation (SD) of at least three independent experiments. Statistical significance was performed using ANOVA analysis to evaluate the significant statistical difference among groups at p < 0.05. ACKNOWLEDGEMENT This research was funded by the National Natural Science Foundation of China (81273459, 81573006), National Basic Research Program of China (2013CB932500), and National Science and Technology Major Project (2012ZX09304004). Supporting Information Experimental details and additional figures (Buffering capacity, TEM, Reductive degradation of RHB and pDNA release study, pDNA condensing ability of RHB and stability of nanoassemblies, Competitive binding assay, and Effects of inhibitors on cellular uptake and transfection efficiency of nanoassemblies). This material is available free of charge via the Internet at http://pubs.acs.org/.


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TOC graphic 381x251mm (96 x 96 DPI)


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Figure 1. The synthetic schemes of RHB. 267x101mm (300 x 300 DPI)



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Figure 2. The 1H NMR spectrums of CBA (A), MBA (B), and RHB (C). 141x135mm (96 x 96 DPI)


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Figure 3. The cytotoxicity, erythrocytes agglutination, and hemolysis evaluation of RHB/pDNA and HA/RHB/pDNA nanoassemblies in vitro. (A) Cytotoxicity of RHB/pDNA at weight ratios of 2.5:1, 5:1, 10:1, 25:1, and 50:1 against B16F10 cells; (B) Cytotoxicity of HA/RHB/pDNA nanoassemblies at weight ratios of 1:5:1, 2.5:5:1, and 5:5:1 against B16F10 cells; (C) Erythrocytes agglutination of RHB/pDNA and HA/RHB/pDNA nanoassemblies. Agglutination was observed by microscopy (200 × magnification). (a) PBS, (b) RHB/pDNA nanoassemblies at weight ratio of 5:1, (c-e) HA/RHB/pDNA nanoassemblies at weight ratios of 1:5:1, 2.5:5:1, and 5:5:1; (D) Hemolytic activity induced by RHB/pDNA nanoassemblies at weight ratios of 2.5:1, 5:1, 10:1, 25:1, and 50:1 in vitro; (E) Hemolytic activity induced by HA/RHB/pDNA nanoassemblies at weight ratios of 1:5:1, 2.5:5:1 and 5:5:1 in vitro. 0.9% saline and Triton X-100 were used as negative control and positive control, respectively. Each bar represents the mean ± SD (n = 5). *: p < 0.05, **: p < 0.01, ***: p < 0.001. 236x90mm (300 x 300 DPI)



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Figure 4. Transfection efficiency of RHB/pDNA nanoassemblies at various weight ratios (2.5:1, 5:1, 10:1, 25:1, and 50:1) in B16F10 cells. LipofectamineTM 2000/pDNA complexes were used as a positive control. (A) Representative fluorescent images of B10F10 cells treated with RHB/pDNA nanoassemblies. Original magnification: 20×. (B) Transfection efficiency of RHB/pDNA nanoassemblies evaluated by flow cytometry. Each bar represents mean ± SD (n=5). **: p < 0.01. 206x132mm (96 x 96 DPI)


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Figure 4. Transfection efficiency of RHB/pDNA nanoassemblies at various weight ratios (2.5:1, 5:1, 10:1, 25:1, and 50:1) in B16F10 cells. LipofectamineTM 2000/pDNA complexes were used as a positive control. (A) Representative fluorescent images of B10F10 cells treated with RHB/pDNA nanoassemblies. Original magnification: 20×. (B) Transfection efficiency of RHB/pDNA nanoassemblies evaluated by flow cytometry. Each bar represents mean ± SD (n=5). **: p < 0.01. 103x91mm (300 x 300 DPI)



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Figure 5. Transfection efficiency of HA/RHB/pDNA nanoassemblies at various weight ratios (0:5:1, 1:5:1, 2.5:5:1, and 5:5:1) in B16F10 and NIH3T3 cells. LipofectamineTM 2000/pDNA complexes were used as a positive control. (A) Representative fluorescent images of B16F10 and NIH3T3 cells treated with HA/RHB/pDNA nanoassemblies. Original magnification: 20×. (B) Transfection efficiency of HA/RHB/pDNA nanoassemblies in B16F10 and NIH3T3 cells quantified by flow cytometry. Each bar represents the mean ± SD (n = 5). *: p < 0.05, ***: p < 0.001. 238x281mm (96 x 96 DPI)


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Figure 5. Transfection efficiency of HA/RHB/pDNA nanoassemblies at various weight ratios (0:5:1, 1:5:1, 2.5:5:1, and 5:5:1) in B16F10 and NIH3T3 cells. LipofectamineTM 2000/pDNA complexes were used as a positive control. (A) Representative fluorescent images of B16F10 and NIH3T3 cells treated with HA/RHB/pDNA nanoassemblies. Original magnification: 20×. (B) Transfection efficiency of HA/RHB/pDNA nanoassemblies in B16F10 and NIH3T3 cells quantified by flow cytometry. Each bar represents the mean ± SD (n = 5). *: p < 0.05, ***: p < 0.001. 125x91mm (300 x 300 DPI)



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Figure 6. Serum-resistant transfection of HA/RHB/pDNA nanoassemblies compared with RHB/pDNA nanoassemblies in vitro. (A) Representative fluorescent images of B16F10 cells treated with (a) RHB/pDNA (w/w, 5:1) nanoassemblies, (b) HA/RHB/pDNA (w/w/w, 5:5:1) nanoassemblies, and (c) LipofectamineTM 2000/pDNA complexes in the presence of 0%, 10%, 20%, and 40% FBS, respectively. Original magnification: 20 ×. (B) The percentage of B16F10 cells expressing GFP determined by flow cytometry. The data were presented as mean ± SD (n = 5). 156x180mm (96 x 96 DPI)


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Figure 6. Serum-resistant transfection of HA/RHB/pDNA nanoassemblies compared with RHB/pDNA nanoassemblies in vitro. (A) Representative fluorescent images of B16F10 cells treated with (a) RHB/pDNA (w/w, 5:1) nanoassemblies, (b) HA/RHB/pDNA (w/w/w, 5:5:1) nanoassemblies, and (c) LipofectamineTM 2000/pDNA complexes in the presence of 0%, 10%, 20%, and 40% FBS, respectively. Original magnification: 20 ×. (B) The percentage of B16F10 cells expressing GFP determined by flow cytometry. The data were presented as mean ± SD (n = 5). 107x76mm (300 x 300 DPI)



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Figure. 7. Intracellular distribution of HA/RHB/pDNA nanoassemblies in B16F10 cells. B16F10 cells were incubated with HA/RHB/YOYO-1-pDNA (green) nanoassemblies. At different time intervals after incubation, cells were stained with Lyso-Tracker Red (red, endosome-lysosome system) and Hoechst33342 (blue, nucleus) and imaged by confocal microscope. Yellow dots in merged images indicated YOYO-1-pDNA (green) trapped within acidic endosomes/lysosomes components (red). 226x163mm (96 x 96 DPI)


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Figure 8. CD44 targeting and cellular uptake of HA/RHB/pDNA nanoassemblies in vivo. C57BL/6 mice were administrated with PBS, naked pDNA, RHB/pDNA nanoassemblies and HA/RHB/pDNA nanoassemblies by intravenous injection, respectively. Lung tissues of C57BL/6 mice were collected at 30 min, 4 h and 24 h post-injection. Frozen slices of lung tissues were sectioned and the fluorescence distribution and cellular uptake of YOYO-1-labeled nanoassemblies in lungs were observed and imaged under fluorescence microscopy. Original magnification: × 20. 431x236mm (96 x 96 DPI)



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Figure 9. The quantitative evaluation of luciferase expression in vivo. The PBS, naked pDNA, RHB/pDNA nanoassemblies, and HA/RHB/pDNA nanoassemblies were injected intravenously into healthy C57BL/6 mice and pulmonary tumor-bearing C57BL/6 mice, respectively. At 48 h after administration, mice were sacrificed and the main organs were dissected to quantify luciferase activity. The luciferase expression is plotted as light units per mg protein (RLU/mg protein). Each bar represents mean ± SD (n = 6). ***: p < 0.001. 201x97mm (300 x 300 DPI)


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Figure 1S. Buffering capacity of RHB obtained by acid–base titration. Titration of RHB was performed in 150 mM NaCl with 0.1M NaOH, and the buffering capacity was analyzed on the pH-titration curves. 123x85mm (300 x 300 DPI)



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Figure 2S. TEM images of the (A) RHB/pDNA and (B) HA/RHB/pDNA nanoassemblies. 136x65mm (96 x 96 DPI)


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Figure 3S. Agarose gel retardation assay of RHB/pDNA nanoassemblies at weight ratios of 2.5:1, 5:1, 10:1, and 50:1 (A) in absence or (B) in presence of DTT (lane 1: naked pDNA; lane 2-5: RHB/pDNA nanoassemblies at weight ratios of 2.5:1, 5:1, 10:1, and 50:1, respectively). 356x118mm (96 x 96 DPI)



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Figure 4S. Agarose gel retardation electrophoresis assay for the formation and stability of RHB/pDNA and HA/RHB/pDNA nanoassemblies in vitro. (A) pDNA condensing ability of RHB at various weight ratios of RHB/pDNA (lane 1: pDNA ladder; lane 2: naked pDNA; lane 3-8: RHB/pDNA nanoassemblies at weight ratios of 1:1, 2.5:1, 5:1, 10:1, 50:1, and 100:1, respectively); (B) The stability of RHB/pDNA nanoassemblies after adding HA (lane 1: pDNA ladder; lane 2: naked pDNA; lane 3: RHB/pDNA nanoassemblies at weight ratio of 5:1; lane 4-6: HA/RHB/pDNA nanoassemblies at weight ratios of 1:5:1, 2.5:5:1, and 5:5:1, respectively); (C) The pDNA protection of nanoassemblies against DNase I at different concentrations (lane 1: pDNA ladder; lane 2: naked pDNA; lane 3, 7, 11: RHB/pDNA nanoassemblies at weight ratio of 5:1; lane 4, 8, 12: HA/RHB/pDNA nanoassemblies at weight ratio of 1:5:1; lane 5, 9, 13: HA/RHB/pDNA nanoassemblies at weight ratio of 2.5:5:1; lane 6, 10, 14: HA/RHB/pDNA nanoassemblies at weight ratio of 5:5:1); (D) The stability of nanoassemblies in sodium heparin (lane 1: pDNA ladder; lane 2: naked pDNA; lane 3, 7, 11: RHB/pDNA nanoassemblies at weight ratio of 5:1; lane 4, 8, 12: HA/RHB/pDNA nanoassemblies at weight ratio of 1:5:1; lane 5, 9, 13: HA/RHB/pDNA nanoassemblies at weight ratio of 2.5:5:1; lane 6, 10, 14: HA/RHB/pDNA nanoassemblies at weight ratio of 5:5:1); (E) The stability of nanoassemblies in serum (lane 1: pDNA ladder; lane 2: naked pDNA; lane 3, 7, 11: RHB/pDNA nanoassemblies at weight ratio of 5:1; lane 4, 8, 12: HA/RHB/pDNA nanoassemblies at weight ratio of 1:5:1; lane 5, 9, 13: HA/RHB/pDNA nanoassemblies at weight ratio of 2.5:5:1; lane 6, 10, 14: HA/RHB/pDNA nanoassemblies at weight ratio of 5:5:1). 410x234mm (96 x 96 DPI)


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Figure 5S. Competitive binding assay. B10F10 and NIH3T3 cells were pre-treated with excess anti-CD44 antibody before transfection with HA/RHB/pDNA nanoassemblies at weight ratio of 5:5:1, respectively. (A) Qualitative fluorescence images and (B) quantitative transfection efficiency of B16F10 and NIH3T3 cells. The data were presented as mean ± SD (n = 5). ***: p < 0.001. 347x121mm (96 x 96 DPI)



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Figure 6S. Effect of different inhibitors on the cellular uptake and transfection efficiency of HA/RHB/pDNA nanoassemblies. (A) Normalized cellular uptake and transfection efficiency of HA/RHB/pDNA nanoassemblies in B16F10 cells pre-treated with endocytosis inhibitors. (B) Normalized transfection efficiency of HA/RHB/pEGFP nanoassemblies in B16F10 cells pre-treated with different inhibitors. B16F10 cells were pretreated with distinct inhibitors for 2 h and then transfected with the nanoassemblies. Mean fluorescence intensity from 10,000 cells was determined by flow cytometry for each sample. Control group was the cells treated with the test nanoassemblies only. The fluorescence intensity in control cells was set to 100%. Each bar represents mean ± SD (n = 3). **: p < 0.01; ***: p < 0.001 compared to control. 251x88mm (300 x 300 DPI)


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CD44-Targeted Hyaluronic Acid-Coated Redox ... - ACS Publications

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