Shear Effects on Stability of DNA Complexes in the Presence of Serum

Aug 21, 2017 - Shear rate dependence of Rh,app, Iex (A), and the corresponding I/R3 value (B) of K20/pDNA/FBS after sheared for 4 h. ...... Van Puyvel...
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Shear Effects on Stability of DNA Complexes in the Presence of Serum Hao Wen, Qiuhong Yu, Yudan Yin, Wei Pan, Shuang Yang,* and Dehai Liang* Beijing National Laboratory for Molecular Sciences and the Key Laboratory of Polymer Chemistry and Physics of Ministry of Education, College of Chemistry and Molecular Engineering, Peking University, Beijing, China, 100871 S Supporting Information *

ABSTRACT: The behavior of nanocarriers, even though they are well-defined at equilibrium conditions, is unpredictable in living system. Using the complexes formed by plasmid DNA (pDNA) and K20 (K: lysine), protamine, or polylysine (PLL) as models, we studied the dynamic behavior of gene carriers in the presence of fetal bovine serum (FBS) and under different shear rates, a condition mimicking the internal physical environment of blood vessels. Without shear, all the positively charged complexes bind to the negatively charged proteins in FBS, leading to the formation of large aggregates and even precipitates. The behaviors are quite different under shear. The shear generates two effects: a mechanical force to break down the complex into smaller size particles above a critical shear rate and a stirring effect leading to secondary aggregation of complexes below the critical shear rate. In the studied shear rate from 100 to 3000 s−1, the mechanical force plays a key role in K20/pDNA and protamine/pDNA, while the stirring effect is dominant in PLL/pDNA. A model study shows that the interfacial tension, the chain density, and the elasticity of the complexes determine their responsiveness to shear force. This study is helpful to understand the fate of drug/gene carriers under physiological conditions. It also gains insight in designing drug/gene carriers with desirable properties for in vivo applications. When administrated intravenously, DNA complexes will first encounter the components in serum. It is reported that the positively charged DNA complexes will form aggregate in serum and be eliminated by phagocytic cells.19−21 In certain cases, the aggregates can further develop into thrombosis.22 Moreover, the complex particles inside the blood vessel also suffer from a shearing force generated by bloodstream. The shear rate of human circulation is ranged from 1 to 105 s−1 depending on the dimension of blood vessels.23 This mechanical force regulates the properties and functions of the cells and proteins in blood. One typical example is the conformational transition of von Willebrand factor (VWF) from globule to stretched conformation at a critical shear rate, which is a key to stop bleeding and execute vascular repairing.23 The soft particles, such as microscale aggregates,24 hydrogel,25 and liposome,26,27 also undergo a conformational change or break down under shear. Different theoretical models have been proposed to predict the steady state deformation, the critical capillary number for breakup, and the breakup mode of a drop in the shear flow as a function of the viscosity ratio and flow type.28−30 Several shear-responsive nanotherapeutics for drugs targeting at obstructed blood vessels have been designed in the past few years.31 However, the effect of shear force on the structure of DNA complex inside the blood vessel has not

1. INTRODUCTION Polycations, such as polylysine, polyethylenimine, and poly(amidoamine), can condense DNA via electrostatic interaction and protect them from degradation under physiological conditions.1 Therefore, the complex particles formed by DNA and polycations have been used as nonviral vectors for gene delivery.2−5 In general, the vectors based on DNA complexes are prepared ex vivo and then administrated intravenously. The ex vivo fabrication of the complex are under close to equilibrium conditions. It is demonstrated that the structure of the complex and the kinetics of complexation are controlled by many parameters, including the nature of polycations,6−8 the mixing ratio9−11 of the complexes, the mixing order,12 and so on. The complexes with well-defined size, shape, and surface charge can be prepared ex vivo by polycations with desirable components at proper conditions. However, the journey and destiny of the complex in vivo is still hard to predict, even though it has been reported that attaching a protecting layer13−16 or ligand17,18 on the surface of the complex is able to prolong the circulation time and achieve targeting on chosen cell. No definite relationship between the physicochemical properties of the complex and its in vivo transfection efficiency has been established so far. The reason lies in the case that the biological systems are never at thermodynamic equilibrium. The metabolism and other life processes, which consume energy and transform matters, also apply to the administrated DNA complexes. © XXXX American Chemical Society

Received: June 28, 2017 Revised: August 18, 2017 Published: August 21, 2017 A

DOI: 10.1021/acs.biomac.7b00900 Biomacromolecules XXXX, XXX, XXX−XXX

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sample preparation and DLS experiment at chosen time point. No filtration is conducted after shear.

been elucidated. The difficulty rests in the detection of DNA complex surrounded by serum in bloodstream. In this work, we use a rheometer with a double-gap Couette to mimic the physiological conditions in bloodstream and apply shear on DNA complex in the presence of serum. Laser light scattering (LLS) is employed to study the size and dynamics of DNA complex after shear treatment. The combination of rheometer and LLS can reveal the effect of shear rate and shear time on the stability of DNA complex. Three different types of polycations, K20 (K: lysine), PLL, and protamine, are chosen to form complex with plasmid DNA (pDNA). The complex sample is mixed with FBS and transferred to the rheometer which offers a precise control on the shear rate and time period of treatment. Results show that the K20/pDNA complex binds to proteins in FBS, forming large aggregates and even precipitates under static condition. However, the aggregate maintains its size or even breaks down in the presence of FBS under shear depending on the treatment time and shear rate. The protamine/pDNA complex in FBS shows similar behavior as K20/pDNA complex, while the PLL/pDNA complex exhibits an opposite trend under the same conditions. It forms secondary aggregates instead of breaking down into smaller size particles.

2. EXPERIMENTAL SECTION

3. RESULTS AND DISCUSSION The DNA complex used for gene delivery is usually prepared at ± ratios larger than unity, in that the electrostatic attraction between the positively charged complex and the cell membrane facilitates the uptake of the complex.32,33 All the polycations, K20, PLL, and protamine, form a complex with pDNA right after mixing in DPBS buffer at studied ± ratios. However, the complexes are not stable in the presence of FBS without shear. Further aggregation and even precipitation occur with time. For example, the hydrodynamic radius (Rh) of K20/pDNA complex at ± = 10 is only ∼280 nm before mixing with FBS (Figure S1). The size increases to the order of micron after being mixed with FBS in 4 h, as revealed by AFM and DLS (Figure S2). The behavior of the complex in the presence of FBS is quite different when shear is applied. Interestingly, the effect of shear on the DNA complex is also dependent on the types of polycations. 3.1. K20/pDNA/FBS under Shear. First, we studied the effect of shear on the behavior of K20/pDNA complex in the presence of FBS. A stable aggregate with Rh about 430 nm is obtained at the shear rate of 100 s−1 after the mixing of K20/ pDNA complex with FBS (Figure 1A). The excess scattered

2.1. Materials and Sample Preparation. Peptide (K20, purity > 98%) was synthesized by purified GL Biochem. Ltd. (Shanghai, China). Dulbecco’s phosphate-buffered saline (DPBS) was purchased from Invitrogen (Shanghai, China). PLL (Mw: 30000−70000 Da) and protamine sulfate (Pro) were purchased form Sigma (Shanghai, China). Fetal bovine serum (FBS) was purchased from M&C Gene Technology Ltd. (Beijing, China) and plasmid DNA (pDNA) was kindly provided by Prof. Quan Du (Peking University). K20 (0.02 g/ L), PLL (0.02 g/L), Pro (0.02 g/L), pDNA (0.1 g/L), and 10% FBS were prepared in DPBS buffer and stored at 4 °C prior to use. Different amounts of pDNA solution was added to K20, PLL, or Pro solution (1.0 mL) to obtain the complex at different ± ratios (±, the molar charge ratio, stands for the number of positively charged amino groups from all the polycations to the number of negatively charged phosphate groups from all the pDNA). The complex sample was gently shaken for 30 s and equilibrated for 30 min. The complex solution was then mixed with same volume of 10% FBS to obtain the complex/FBS aggregates. Each reagent including FBS is filtered to remove dust before mixing. 2.2. Dynamic Light Scattering (DLS). In dynamic light scattering (DLS), the intensity−intensity time correlation function G(2)(τ) in the self-beating mode was measured. A Laplace inversion program, CONTIN, was used to process the data to obtain the line width distribution and diffusion coefficient. The diffusion coefficient D can be further converted into the hydrodynamic radius Rh by using the Stokes−Einstein equation:

Figure 1. Shear time dependence of Rh,app (A), Iex (B), and size distribution (C, D) of K20/pDNA/FBS aggregates. The smaller size fragments in (C) and (D) are attributed to protein or associates of protein in FBS. Shear rate: 100 s−1 (C) and 3000 s−1 (D). Scattered angle: 30°.

D = (kBT )/(6πηR h)

(1)

intensity Iex (denoted as (Is − I0)/It, with Is, I0, and It being the scattered intensity from the complex solution, the solvent, and toluene, respectively) of K20/pDNA/FBS keeps unchanged (Figure 1B). The time dependence of size distribution (Figure 1C) and morphology (Figure 2A) also confirms that the aggregate is stable after being treated at 100 s−1. At 3000 s−1, however, a remarkable change in size is observed after being sheared for 2 h. The Rh value of the aggregate significantly drops from 470 to 240 nm (Figure 1A), which is also revealed by the change in size distribution (Figure 1D) and AFM images at 3000 s−1(Figure 2B). On the contrary, the Iex increases (Figure 1B), indicating that the initially formed aggregates are broken down into smaller but more compact particles. Leal et

where kB, T, and η are the Boltzmann constant, the absolute temperature, and the viscosity of the solvent, respectively. 2.3. Atomic Force Microscopy (AFM). The AFM images were recorded using a Dimension Icon Atomic Force Microscope (Brüker, U.S.A.). A total of 30 μL of the sample solution was deposited and kept standing for 40 s on a fresh mica surface. The excess solution was then blotted away with a strip of filter paper, and the mica surface was washed with 50 μL of deionized water to remove the salt. The sample was air-dried for 1 day before AFM experiments. 2.4. Polyelectrolyte Complexes/FBS Aggregates under Shear. Shear stress was generated by MCR301 rheometer (Anton Paar, Graz, Austria) equipped with double-gap Couette geometry. The mixture of DNA complex and FBS solution was loaded to the Couette and treated at chosen shear rate. The sample was taken out for AFM B

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Figure 2. AFM images and height profile of selected particles (inset) of K20/pDNA/FBS aggregates before and after shear at 100 s−1 (A) and 3000 s−1 (B) for different shear time. Scale bar: 1 μm.

Figure 3. Shear rate dependence of Rh,app, Iex (A), and the corresponding I/R3 value (B) of K20/pDNA/FBS after sheared for 4 h. The K20/pDNA charge ratio is 10 (scattered angle: 30°).

Figure 4. AFM images and the corresponding height profile (inset) of selected K20/pDNA/FBS aggregates right after mixing without shear (A) and after being treated at a shear rate of 100 (B), 500 (C), 1000 (D), 3000 (E), and 5000 s−1 (F) for 4 h. Scale bar: 1 μm.

al.34 reported that the breakup of a drop containing surfactants or copolymers in shear flow is accompanied by a redistribution of the molecules, leading to an increase in interfacial tension of the large drop and a decrease in interfacial tension of daughter drops. Redistribution of molecules could also occur as the K20/ pDNA/FBS aggregates breakup in our study. As the content of hydrophobic component becomes higher, the interfacial tension of the K20/pDNA/FBS droplet increases after shear.

These more compact K20/pDNA/FBS droplets generate a strong scattered intensity in spite of the decrease in size. The behavior of the complex treated at other shear rates for 4 h is shown in Figure 3. The Rh of the complex gradually decreases with increasing shear rate, and then becomes stable as the shear rate reaches 1000 s−1. No prominent change in size distribution (polydisperisty) is observed at the studied shear rates (Figure S3). The dependence of Iex on shear rate is, C

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Biomacromolecules however, complicated due to a sharp increase at 500 s−1 (Figure 3A). At fixed concentrations, the temporal chain density of the complex (without considering the contribution from the solvent) can be evaluated by Iex/R3. Figure 3B shows that the chain density of the aggregate only slightly increases at 100 and 300 s−1. A significant jump is observed at 500 and 1000 s−1, which explains the changes in Iex. The significant jump of chain density also indicates a more compact K20/pDNA/FBS aggregates caused by the material redistribution as mentioned above. At shear rate above 1000 s−1, the chain density becomes stable. The changes in morphology and size of K20/pDNA complex in the presence of FBS treated at different shear rates are also determined by AFM (Figure 4). Mainly spherical particles are observed at the studied conditions, especially when the shear rates are low. The complexes form large aggregates right after mixing, as indicated by the particle with the height about 200 nm (Figure 4A). After being treated at 100 or 500 s−1 for 4 h, the aggregate maintains its size (Figure 4B,C). However, the sizes of the aggregates are much smaller after being treated at higher shear rates. The height of the particles also decreases with increasing shear rate (Figure 4D−F) It is known that the hydrodynamic stress induced by the flow on the drop surface tends to deform and breakup the drop, and the interfacial stress between the phases resists this deformation.28,35 Different theoretical models28,29 are introduced to predict of the steady state deformation and the critical capillary number for breakup, but few of them could predict the final drop size or size distribution after breakup. Herein, we build a simple model to explain the obtained results, which may also predict the relationship between the surface tension and the final size after shear. The formation of complex particle by oppositely charged polyelectrolytes is driven by electrostatic interactions. The neutralization of charges results in lower dielectric constant and higher interfacial tension.36,37 The complex particle is thus in a spherical shape (Figure 5A), and its

The center of the sphere is set as the origin O. When the sphere rotates at an angular velocity ω along an axes across its center, the most unstable part is the equator region, which is perpendicular to the axes and has the largest cross section (Figure 5A). After losing some content under spinning, the radius of the sphere becomes R. For a volume element Δν = Δs·dr (Figure 5A), the pressure difference dP between its inner and outer surfaces provide the balanced force against the centrifugal force dP·Δs = Δmrw2, where the mass of volume element is Δm = ρ̅ ·Δs·dr (ρ̅ is the averaged mass density). The pressure difference can thus be written as dP = ρ̅rω2dr. By integration:

∫P

Ps

0

dP = Ps − P0 =

∫0

R

ρ ̅ rω 2dr = ρ ̅ R2ω 2 /2

(2)

On the out surface, the pressure difference needs to satisfy Young−Laplace equation ΔP = Ps − Pout = σ/2R. Considering ΔP0 = P0 − Pout = σ/2R0, the new particle size satisfies: σ(R 0 − R ) = ρ ̅ R 0w 2R3

(3)

For a rigid sphere suspended in a Newtonian fluid with shear rate γ̇, its rotation period T is given by T = 4π/γ̇,38 and the angular velocity ω follows ω = 2π/T = γ̇/2. The linear relationship is hold only at small shear Reynolds number,39 and it can be applied to our system safely. Equation 3 then becomes 4σ(R 0 − R ) = ρ ̅ R 0γ 2̇ R3

(4)

Equation 4 is then used to describe the experimental results. As shown in Figure 5B, this model fits the data well at γ̇ < 1000 s−1. At higher γ̇ values, the particle size is basically independent of the shear rate. One possibility is that the material distribution during deformation and breakup of the K20/pDNA/FBS aggregates leads to a higher content of component in K20/ pDNA/FBS droplets, leading to an increase in the viscosity as well as interfacial tension of the droplet. However, a critical viscosity ratio (∼3.6) between the drop and solvent is predict by small deformation theory and experimentally verified in simple flow,28,40,41 above which the drop breakup is impossible. It is probably that the viscosity ratio between the K20/pDNA/ FBS droplets and water exceeds the critical value at the shear rate above 1000 s−1. The other possibility is the limit determined by the size of single pDNA. The radius of gyration of pDNA can be estimated by ⟨R2DNA⟩ = Lc·lp/3.42 For pDNA in aqueous solution, its persistent length lp is 50 nm and its counter length is Lc ≈ 1700 nm. Accordingly, the coil size of pDNA is about 170 nm. Taking into account its adsorbed content, this value is very close to the size value (200 nm) of K20/pDNA/FBS aggregates at shear rate above 1000 s−1. It has been demonstrated that charge ratio is a key parameter affecting the stability, cytotoxicity, and efficacy of nonviral vectors.43,44 In this study, we also compared the behavior of K20/pDNA complexes at ± = 2, 10, 20, and 50 in the presence of FBS under shear. As shown in Figure 6A, the size of the complexes formed by K20 and pDNA alone decreases with increasing ± ratio. A similar trend is observed when the corresponding complex is mixed with FBS, separately, before shear. The AFM images (Figure 5) and the excess scattered intensity (Figure S4) also confirm that the complex particle is smaller in size and lower in molecular weight (which is proportional to the excess scattered intensity) at higher charged ratios away from unity. All these results are typical behavior of polyelectrolyte complexes.6,45 However, things are quite

Figure 5. (A) Schematically showing the model under shear, (B) the fitting of the model with the experimental result on the size of K20/ pDNA/FBS aggregates at γ̇ < 1000 s−1 by using eq 3. R2 = 0.9865.

integrity is preserved by the balance between the interfacial tension and the inner pressure. When being sheared by the flow force, the particle rotates freely. The momenta of inertia produce additional pressure, breaking down its balance with the interfacial tension. As a result, the particle will lose some of its content to reach a new equilibrium. For a stationary spherical particle with radius of R0, the interfacial tension σ and the pressure difference ΔP0 satisfies the Young−Laplace equation ΔP0 = P0 − Pout = σ/2R0, where P0 and Pout are the pressures inside and outside of the surface. D

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Figure 6. Rh,app (A) and corresponding I/R3 value (B) of K20/pDNA complex and K20/pDNA/FBS aggregates at different ± ratios before and after being sheared at 3000 s−1 for 4 h (scattered angle: 30°).

Figure 7. AFM images and the corresponding height profiles of selected particles of K20/pDNA/FBS aggregates at different ± ratios before (A−D) and after (E−H) being sheared at 3000 s−1 for 4 h. Scale bar: 1 μm.

Figure 8. Rh,app (A) and Iex (B) of Pro/pDNA/FBS before and after sheared for 4 h at different shear rates. The Pro/pDNA charge ratio is 10. Scattered angle: 30°.

different after the complexes are sheared at 3000 s−1 for 4 h. Both LLS (Figure 6A and S5) and AFM (Figure 7) show that the size or height of the complex particle at ± ≤ 20 significantly decreases, suggesting that the aggregates are broken down by shear. The Iex/R3 values of the particle after being treatment are also calculated using the data on size (Figure 6A) and on excess scattered intensity (Figure S4). As shown in Figure 6B, the temporal chain density of the particles also increases after shear, indicating that the aggregates at ± ≤ 20 are broken down into more compact particles. However, the final sizes of the particle at ± = 10 and 20 after shear are obviously smaller than that of the particle at ± = 2. It is known that the complex formed at charge ratios far from unity is generally more hydrophilic due to the un-neutralized charges on the surface.10 Such complex possess a lower interfacial tension, and easier to break down under shear

according to eq 4. The particle at ± = 50, which contains large amount of excess polycations, is a special case. The excess polycations form secondary aggregation with FBS as the original particles break down, which is demonstrated by the increased size (Figure 4A and S5) and relative low chain density. We attribute it to the stirring effect of shear, which facilitates the mixing of K20 and FBS. 3.2. Pro/pDNA/FBS under Shear. To test whether the findings based on K20 is applicable to other polycations, we studied the behavior of the complex formed by protamine and DNA under the same conditions. Protamine consists of about 30 amino acid residues, 2/3 of which are arginine. Therefore, it has almost the same amount of charges but a larger molecular weight compared with K20. Figure 8 shows the size and Iex of Pro/pDNA complex (± = 10) treated for 4 h at selected shear rates in the presence of FBS. The Rh decreases with increasing E

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Biomacromolecules shear rate (Figures 8A and S6), while the Iex uprises sharply at 3000 s−1, below which it is relatively stable (Figure 8B). The trends of both Rh and Iex are similar to those in K20/pDNA/ FBS system, suggesting that the complex is able to maintain the structure at lower shear rate, but broken into smaller and more compact particles as the shear rate is beyond certain value. The AFM images (Figure 9) confirm this conclusion. Moreover, eq 4 can also fit the Rh data fairly well (Figure 8A).

distort and break Pro/pDNA/FBS aggregates into smaller pieces. 3.3. PLL/pDNA/FBS under Shear. The complex formed by long chain polycation possesses a higher elasticity. If the elasticity is higher than the mechanical force generated by shear, the complex should not be broken down. To test this hypothesis, we choose PLL, whose length is about 20× that of K20, and studied the behavior of its complex with pDNA under shear. Figure 10 shows the results of PLL/pDNA complex at ± = 10 after being treated at selected shear rate in the presence of FBS. Both the size (Figure 10A) and the excess scattered intensity (Figure 10B) monotonously increases with shear rate after being treated for 4 h, suggesting that the complex further forms aggregates, instead of breaking down under shear. The apparent chain density (Figure S7) of the aggregates almost unchanged after treated at different shear rates, indicating that the aggregates maintained their structure under shear. The size of the aggregates is larger at higher shear rate as detected by LLS, due to the stronger stirring and homogenizing effects under higher shear rate. AFM images (Figure 11) and Rh distribution (Figure S8) also confirm that the size of the PLL/ pDNA/FBS aggregates increases prominently after shear.

Figure 9. AFM images of selected particles of Pro/pDNA/FBS aggregates before (A) and after sheared at 500 (B), 1000 (C), and 3000 s−1 (D) for 4 h. The Pro/pDNA charge ratio is 10. Scale bar: 1 μm.

The critical shear rate above which Pro/pDNA/FBS aggregate breaks into smaller pieces is more than 1000 s−1 (Figure 8), larger than the value (above 500 s−1) for K20/ pDNA/FBS aggregate under the same conditions, suggesting that the nature of the polycation determines the behavior of complex under shear. According to our model in Figure 5, the balance between interfacial tension and the inner pressure of the complex is rebuilt after the distortion and release of content under shear. The distortion and break down of the complex under shear is directly related to its elasticity. Polycations with a longer chain length and stronger affinity for DNA molecules form a complex with a higher elasticity, which can withhold higher shear force. Protamine is longer than K20 and possesses hydrophobic groups. Therefore, a higher shear rate is needed to

Figure 11. AFM images of selected particles of PLL/pDNA/FBS aggregates before (A) and after sheared at 500 (B), 1000 (C), and 3000 s−1 (D) for 4 h. The PLL/pDNA charge ratio is 10. Scale bar: 1 μm.

Figure 10. Rh,app (A) and Iex (B) of PLL/pDNA/FBS before and after sheared for 4 h at different shear rates. The PLL/pDNA charge ratio is 10 (scattered angle: 30°). F

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It is known that the complexation of DNA and polycations are controlled by kinetics.46,47 Both DNA and polycations are not fully neutralized upon from complex. The situation is more severe when the polycation has a longer chain length. Not only that the polycations themselves have a longer relaxation time, the heavy entanglements among the polymer chains also significantly confine each polymer chain in a limited “Tube”.48 On one hand, the enhanced modulus prevents the complex from breaking down under finite shear rate. On the other hand, the complex contains many more un-neutralized loops than that of the complex formed by polyelectrolytes of shorter chains. Therefore, the shear homogenizes the PLL/pDNA complex, leading to a further aggregation in the presence of FBS.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biomac.7b00900. AFM and LLS results on the complex at different shear rate and shear time (PDF).

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REFERENCES

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4. CONCLUSIONS All the model gene carriers undergo a dynamic process in the presence of FBS and under shear, a condition mimicking the internal physical environment of blood vessels. The shear generates two effects: a mechanical force to break down the complexes and a stirring effect to make the complex and the system more homogeneous. The former effect results in smaller size complex particles, while the latter one leads to secondary aggregation of complex. For a chosen gene carrier, there exists a critical shear rate, above which the carrier will break down into smaller pieces. The stirring effect is dominant below the critical shear rate. The responsiveness of the carrier to shear rate is controlled mainly by its interfacial tension, which is determined by the charge ratio, chain length, and other factors. Since the blood flow rate ranges from 1 to 105 s−1 and is specific in chosen organs, our finding offers a new approach to design gene carriers to achieve targeting and controlled release on the basis of physiological regulations.

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AUTHOR INFORMATION

Corresponding Authors

*Tel.: +86-10-62756170. Fax: 86-10-62751708. E-mail: [email protected]. *Tel.: +86-10-62754079. Fax: 86-10-62751708. E-mail: [email protected]. ORCID

Dehai Liang: 0000-0003-4246-050X Funding

Financial support of this work from the National Natural Science Foundation of China (21574002) and Beijing Natural Science Foundation (2171001) is gratefully acknowledged. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank Dr. Quan Du for kindly providing of plasmid DNA. G

DOI: 10.1021/acs.biomac.7b00900 Biomacromolecules XXXX, XXX, XXX−XXX

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DOI: 10.1021/acs.biomac.7b00900 Biomacromolecules XXXX, XXX, XXX−XXX