Albumin-Modified Cationic Nanocarriers To Potentially Create a New

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Biological and Medical Applications of Materials and Interfaces

Albumin modified cationic nanocarriers to potentially create a new platform for drug delivery systems Zhicheng Pan, Xueling He, Nijia Song, Danxuan Fang, Zhen Li, Jiehua Li, Feng Luo, Jianshu Li, Hong Tan, and Qiang Fu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b05599 • Publication Date (Web): 17 Apr 2019 Downloaded from http://pubs.acs.org on April 17, 2019

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ACS Applied Materials & Interfaces

Albumin modified cationic nanocarriers to potentially create a new platform for drug delivery systems Zhicheng Pan1, 3, Xueling He1, 2, Nijia Song1, Danxuan Fang1, Zhen Li1*, Jiehua Li1, Feng Luo1, Jianshu Li1, Hong Tan1* and Qiang Fu1

1College

of Polymer Science and Engineering, State Key Laboratory of Polymer

Materials Engineering, Sichuan University, Chengdu 610065, China

2Laboratory

Animal Center of Sichuan University, Chengdu 610041, China

3Department

of Chemical Engineering, McMaster University, 1280 Main Street West,

Hamilton, Ontario, Canada L8S 4L8

ACS Paragon Plus Environment

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*Corresponding

author. Fax: +86-28-85405402; [email protected], [email protected]

Tel:

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+86-28-85460972;

E-mail:

Keywords: cationic micelles, albumin, multilayer corona, denature, biosafety

ABSTRACT

Cationic nanocarriers have emerged as promising nanoparticles systems for the effective delivery of nucleic acid and anticancer drugs to cancer cells. Positive charge is desirable for promoting cell internalization, while it also causes some adverse effects, such as toxicity and rapid clearance by mononuclear phagocytic systems. Herein, a new strategy of modifying cationic polymer micelles with albumin forming a protein corona to improve the surface physiochemical properties is reported. The corona with monolayer or multilayer was constructed depending on the albumin concentration, and the proteins


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would denature in different degrees due to the interaction with the surface of cationic micelles. It is demonstrated that multilayer albumin corona is beneficial to prevent macrophages uptake, increase accumulation in tumor tissue and reduce toxic side effects to normal tissue. Our work provides a promising method to modify cationic nanopaltform with optimizing the biosecurity and bioavailability for potential application in drug delivery.


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1. Introduction In recent years, the rapid development of nanomaterials offered a great application potential to improve the treatment and diagnosis of disease. Many smarter nanocarriers were fabricated through sophisticated molecule structure design and integration of multifunctional groups. Diversification of functions and nanoscale structure confer nanocarriers a very large surface-to-volume ratio and undesirable surface properties, like hydrophobicity and high charge density which leading to some adverse effects in vivo. The nanocarriers surface will be immediately covered by hundreds of plasma proteins immediately after entering a physiological environment, forming a cloud of aggregated proteins known as “protein corona”, which endues nanocarriers a new biological identity different from their initial synthetic identity.1 The new ‘‘biological identity’’ will transform the biodistribution of the nanocarriers as well as their pathophysiological and therapeutic effects.2-4 Among the protein corona, immunoglobulins (IgG) and complement proteins,5 called opsonin proteins, are known to associate with mononuclear phagocytic systems (MPS), which will promote recognized and subsequent elimination of nanocarriers.6 In


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addition, the adsorbed proteins of nanocarries can activate the enzymatic cascade processes which will lead to thrombosis or anaphylaxis.7 Therefore, elimination of the undesirable protein adsorption of nanocarries plays a key role in improving the biocompatibility and therapy effect.8 Unexpectedly, some proteins display ideal raw materials for drug delivery, since they have the natural advantages over synthetic polymers, such as the biodegradability, metabolism and low toxicity.9-11 For instance, albumin as the most abundant plasma protein has been widely used as a versatile protein vehicle for drug delivery system, due to its excellent biocompatibility, inexpensive production, lack of immunogenicity and toxicity, as well as easy storage and biostability.11-13 Albumin nanocarriers display high binding capacity of various drugs and targeting ligands owing to conjugated abundant functional groups (carboxylic groups or amino groups) on the surfaces.14-15 Meanwhile, Albumin nanoparticles can incorporate a large variety of drugs via electrostatic interaction or hydrophobic interaction.16 Abraxane®, an albumin-drug nanocarrier formed by the hydrophobic interaction between human serum albumin and paclitaxel (PTX), has been approved by the US Food and Drug Administration (FDA) and becomes a first-line anti-


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cancer drug for breast, lung and pancreatic cancer.17-18 Moreover, the albumin nanoparticles could accumulate in the tumor through transport pathway mediated by the 60-kDa glycoprotein (gp60), which is an albumin-binding glycoprotein expressed in endothelial cells around tumors.19-22 Cationic nanocarriers have emerged as promising nanoparticles systems for the effective delivery of nucleic acid and anticancer drugs to cancer cells. Positive charge is desirable as it enhances the cell uptake due to the charge attraction, thereby increasing the rate and extent of internalization.23 However, positive surface charge also causes some adverse effects including toxicity, protein adsorption and rapid clearance by the MPS.24 On the one hand, biologically inert surface of nanocarriers was strived to design and construct to prevent the unfriendly plasma protein adsorption. For another, some proteins, like Albumin, were used as ideal drug carriers for clinic cancer treatment. Therefore, we combine these two seemingly contradictory things to create a new nanoplatform for cancer treatment. In our previously study, a facile and versatile approach was provided for fabrication of smart intracellular targeted polyurethane micelles for effective tumor treatment.25-28 We


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have shown that gemini quaternary ammonium (GQA), as a cell internalization promoter, can strengthen the permeability of polyurethane micelles and enhance the cellular uptake.29 However, we also have found that GQA was hardly completely shielded by the polyethylene glycol (PEG) chains.30 The exposed cationic GQA might puncture the cell membrane, damage the integrity of cell and potentially cause apoptosis. Moreover, GQA causes amounts of protein absorption on the polyurethane micelles via electrostatic interaction.31 In order to eliminate the adverse protein absorption and improve biosecurity, albumin was primarily utilized to modify the cationic polyurethane micelles to obtain a micelles-albumin complex (MAC). Depending on the concentration of the albumin, the adsorbed proteins form corona with monolayer or multilayers surrounding the micelles, and the closer to the surface of micelles, the albumin denaturation appears more severe. Due to the different degree of protein denaturation, MAC with single or multilayer albumin corona display different physiochemical properties on surface and biological properties in

vitro, especially the internalization by tumor cells and escape from macrophage. Moreover, the albumin corona served as a protective shell for cationic micelles, which could prevent the opsonin proteins adsorption during blood circulation and relieve the


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elimination by reticuloendothelial system (RES) in vivo. The micelles-albumin complex could effectively improve the biosecurity of cationic micelles without affecting the treatment of tumors. This work aim to provide a new method to effectively improve the biodistribution and biosecurity for cationic nano-platform using in the drug delivery system.

2. Experimental Section 2.1 Materials The cationic polyurethane micelles with gemini quaternary ammonium were prepared according to previous reports.32 The anticancer drug Taxol and PTX was purchased from Shanghai Jinhe Bio-Technology Co. A dialysis bag was purchased from Solarbio (MWCO 3.5 kDa). Fluorescein isothiocyanate isomer I (FITC, 90%) and 2-(4-Amidinophenyl)-6indolecarbamidine dihydrochloride (DAPI) was purchased from Acros Organics, USA and Roche Diagnostics, Germany, respectively. CCK-8 (Cell Counting Kit-8) was obtained from Dojindo, Japan. Mouse serum albumin (MSA) and bovine serum albumin (BSA) was purchased from Sigma. The 0.45 μm pore-sized syringe filter was obtained from Milipore, Carrigtwohill, Co. Cork, Ireland.


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2.2 Preparation of cationic polyurethane micelles and MACs The polyurethane of G8mE1900 (50 mg) was completely dissolved in 10 ml N, Ndimethylacetamide (DMAc). The polymer solution was added dropwise to deionized water (30 ml). After that, the resulting solution was dialyzed (MWCO = 3500 g mol−1) against deionized water for a minimum of 6 cycles at room temperature. Finally the micelles was obtained by passed through the 0.45 μm syringe filter. MACs was prepared by mixed the naked G8mE1900 micelles and different concentration albumin with gentle stirring for 4 h at 25 oC. Then the resulting solution passed through the 0.45 μm syringe filter to obtain the MACs solution. To avoid the mouse immune response caused by bovine serum albumin, all the MACs was prepared by mouse serum albumin (MSA) for the in vivo experiments. 2.3 Characterization of naked G8mE1900 micelles and MACs The size, size distribution and zeta potential of naked G8mE1900 micelles and MACs were measured by DLS, the morphology was observed by transmission electron microscopy as described previously31.


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Accurate BSA (50 µg/ml) with naked G8mE1900 micelles (25 or 500 µg/ml) concentrations were absolutely prepared in water. The secondary structure properties of BSA proteins was measured by Circular dichroism (CD). A quartz cuvette (0.1 cm pathlength) was used and data were collected from 180 to 300 nm at 25 °C, deionized water was choose as the background. 2.4 Characterization of cellular uptake The internalization behavior and intracellular distribution of the naked G8mE1900 and MACs were observed using confocal laser scanning microscopy (CLSM) and flow cytometry microscopy as described previously26. Briefly, HeLa and Raw264.7 cells were plated at a density of 1×105 cells/well in a 6-well plate and maintained in 1640 media supplemented with 10% FBS and 1% penicillin. FITC labeled naked G8mE1900 micelles and MACs by physical entrapment. Then, the fluorescent labeled micelles were transferred into wells with cultured cells and incubated for 30 min and 60 min. After that, each well was washed three times with 0.1 M PBS. For CLSM, the 4% paraformaldehyde was added into each well to fix the cell for 30 min. The cell was stained by DAPI for 10 min at room temperature. For flow cytometry, followed by trypsin treated and


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centrifugation to collect the cells. Then removing the supernatant, using in 0.5 mL 0.1 M PBS to resuspend cell, and analyzed by Cytomics FC 500 (Beckman Coulter). 2.5 Cytotoxicity asssy The cytotoxicity of naked G8mE1900 micelles and MACs were evaluated employing HeLa cells with in vitro proliferation via the CCK-8 cell viability assay kit. The HeLa cells were plated at a density of 5 000 cells/well in a 96-well plate. Drug-loaded or drug-free naked G8mE1900 micelles and MACs with various concentrations were transferred into wells with cultured cells and incubated for 24 h and 72 h. Cell viability was then characterised using a CCK-8 assay kit30. 2.6 Animals Male nude mice (15 ± 1 g) were obtained from Laboratory Animal Center of Sichuan University (Sichuan, China). They were free to access the standard food and water in a light controlled room at 25± 2 ºC with the humidity of 55 ± 5%. The animal procedures were performed according to were approved by the Ethics Committee of Sichuan University. The Principles of Laboratory Animal Care of the National Institutes of Health for the use and care of laboratory animals were also confirmed.


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2.7 In vivo tumor growth and biodistribution studies To assess the tissue distribution of MACs in vivo, Hela tumor-bearing mice were injected with FITC-labeled MACs and naked G8mE1900 micelles at dosage of 30 mg/ml (FITC was 30%, w/w). The liver, heart, lung, kidney, spleen and tumor were collected from mice after treatment at 60 min and 360 min. The semi-quantitative analysis of the fluorescence intensity at the tumor site was also carried out. 2.8 In vivo antitumor activity The detail of evaluating the antitumor activity in vivo was described previously26. Briefly, the mice were randomly separated (n = 5) into night groups. The tumor which were induced in right back after inoculation of 4 × 106 cells grown to 40 mm3. PTX-loaded naked G8mE1900 micelles, MACs and Taxol (5 mg/kg equivalent PTX) were injected. The control group was received intravenous administration of albumin, saline and blank cationic micelles and MACs. All group were received intravenous injection every three days for five times during 21 days. The tumor volume were measured and calculated every 3 days following the formula: (width2 × length)/2. The survival rate and body weight


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were also recorded every 3 days. After 21 days, the tumors were collected and weighted from sacrificed mice. The tumor and normal tissues were sectioned for H&E staining. 2.9 Liver function test After 21 days injection, the blood samples of each mice (~0.8 mL) were collected. The blood samples were centrifuged (3000 r min−1, 4 ºC) for 10 min. The function markers of liver were added to assess the alanine aminotransferase (ALT). All these samples were measured by IDEXX Biochemical Analyzer (Westbrook, ME, USA) using the kits according to the manufacturer's instructions. 2.10 Statistical Analysis The significance of difference was analyzed by the Social Sciences (SPSS, version 19) software. The details were described as previously reported.25

3. Results and discussion 3.1 Preparation and characterization of polyurethane micelles albumin complex (MAC). Polyurethane micelles bearing GQA have shown high positive charge and high protein adsorption,30 which would reduce the circulation time in blood and therapeutic effect in

vivo. Considering the high non-specific protein adsorption of cationic micelles and the


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advantages of albumin, polyurethane micelles bearing GQA (G8mE1900,31 naked micelles) were cultivated with a range of different concentrations of albumin (BSA) solution to prepare the micelles-albumin complexes (MACs). The size, size distribution, zeta potential and morphology of MAC were examined by DLS and TEM. As shown in Figure 1 and Table S1, the size and zeta potential of naked G8mE1900 micelles are 75 nm and 31 mv, respectively. BSA solution with low concentration (≤ 0.05 mg/ml) could not affect the size of MACs, and the particle size and size distribution stably remain at initial values. However, the zeta potential slowly lower with the concentration increased, since the negative charge of BSA could neutralize a part of the positive charge of GQA. The above data indicated that the BSA at low concentration cannot form a whole protein corona to completely cover the surface of the micelles. When the concentration of BSA reach to 0.1 mg/ml, the size has greatly increased and the zeta potential has a drastic drop. The size and zeta potential were 137 nm and 3 mv, which increased by 78% and decreased by 86% compared with naked micelles, respectively. Meanwhile, the PDI of MACs also has a great change. These phenomena indicate that the micelles albumin complexes were formed when the albumin concentration over 0.1 mg/ml.


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As the protein concentration continues to increase, the zeta potential of MACs has reversed from positive to negative, and the size increase to 172 nm and then drop to 101 nm. In fact, the diameter of MACs keeps increasing until the MAC reached the maximum protein adsorption. Moreover, the higher BSA concentration will pull down the average size of the MACs, as well as the PDI, caused by the smaller size of BSA particle (30~50 nm, Figure 2B). 3.2 The Morphology and Protein Denaturation of the Micelles-Albumin Complex The formation of MACs can be further demonstrated by TEM due to the morphology changed. As shown in Figure 2A, the naked G8mE1900 micelles distribute individually with regular and spherical shape. The diameters of the naked G8mE1900 micelles calculated from TEM is in the range of 70~90 nm, which is similar to the data obtained by DLS. BSA protein also displays regular sphere with the smaller size range of 30-50 nm (Figure 2B). Meanwhile, the morphology has a great change when the albumin concentration reaches to 0.1 mg/ml. As shown in Figure 2C, an adsorbed protein layer surrounding the micellar surface can be obviously observed. The size of G8mE1900-LB (naked G8mE1900 micelles modified by low albumin concentration) has a slight


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increased with irregular spherical shape. Compared to the naked micelles, the size of G8mE1900-LB has exceeded ~50 nm, which is exactly the diameter of a single BSA. Therefore, it can be inferred that the MACs were coated with a monolayer protein corona under 0.1 mg/ml BSA. As the concentration of BSA reaches to 2 mg/ml, the size of G8mE1900-HB (naked G8mE1900 micelles modified by high albumin concentration) expanded to 150~200 nm, owing to more proteins are adsorbed on the micellar surface to construct multilayer protein corona. These distinguishing TEM images provide strong visualized evidence that the formation of monolayer or multilayer albumin corona. According to the results, MACs have received variational particle size and morphology, but still maintain the potential of becoming nanocarriers. In addition, the conversion of surface charge is more favorable to internal circulation. To further study the interaction between cationic micelles and BSA proteins, the secondary structure transformation of BSA proteins was examined by CD spectroscopy. The negative peaks at 208 nm and 222 nm, as well as the maximum positive peak at 192 nm, represent the α-helix structure, and the negative peak at 218 nm belongs to the βsheet structure.33 To compare the changes in albumin secondary structure, the BSA


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concentration was fixed while the micelles concentration were varied. As shown in Figure 3, the absorption at 192 nm has dramatically decreased due to the increased concentration of naked micelles, which gives a good indication that the α-helix structure of BSA protein can be destroyed by the interaction with cationic micelles. Moreover, under the same protein concentration, the higher concentration of naked micelles, the more severe degree of denaturation. Given that the albumin is distributed in multilayer surrounding the micelles, it can be speculated that the closer to the surface of micelles, the greater damage to the secondary structure of BSA protein. Denatured albumin corona is easily recognized and quickly cleared by the immune system, while unaffected one can help cationic nanocarries escape the capture of MPS and increase the circulation time in blood. 3.3 Phagocytosis of Micelles-Albumin Complex by Mouse Macrophages Raw264.7 Cells Phagocytosis, the process by which macrophages engulf foreign particles, is an important pathway to eliminate the exogenous nanocarries from the blood.34-35 Therefore, it is of great importance to examine the impact of albumin corona on the phagocytosis of MACs. To this end, the Raw264.7 cell line treated with the serum-free medium in advance


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is applied to co-culture with MACs. As shown in Figure 4 and Figure S4, it was found that macrophages cultured with MACs exhibit remarkable green fluorescence signal in 0.5 and 1 h. Moreover, the fluorescence signal of G8mE1900-LB was stronger than naked micelles verified by flow cytometry at 1 h. However, the fluorescence signal of G8mE1900-HB was hardly observed both in 0.5 h and 1 h. These results are closely related to the albumin corona. It is well known that macrophages could recognize and eliminate oxidized lipids, denatured proteins, advanced glycation end-products and dead cells.36 Cationic micelles modified by low concentration BSA could destroy the secondary structure to denature the albumin corona, which promoted the phagocytosis of Raw264.7. It should be noted that the zeta potential of G8mE1900-LB is near neutral (3 mv), so that the phagocytosis is only related to the denatured albumin corona, not the electrostatic interaction. While, G8mE1900-HB are endowed with multilayer albumin corona, of which the outer layer is nondestructive native protein and become a steric barrier to prevent macrophages uptake.37 These results indicate that pre-coating the albumin corona has a great potential for reducing the phagocytosis and enhancing the lifetime of cationic nanocarries in vivo.


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3.4 In Vivo Tissue distribution of MAC To further verify the ability of albumin corona to evade the immune system in vivo, the biodistribution of MACs in HeLa tumor-bearing nude mice was imaged by a macro imaging system. The tumor-bearing mice were injected with FITC-loaded naked G8mE190 micelles and MACs, respectively. In Figure 5A, it could be found that the naked micelles have a higher accumulation in normal organs, especially in liver and kidney. In contrast, the fluorescence intensity of G8mE1900-HB dramatically decreased by 26% and 22% in liver and kidney, respectively (Figure 5B). The result indicates that the nondestructive albumin corona can provide good protection for cationic nanocarriers and effectively suppress the recognition and subsequent elimination by RES system. In addition, G8mE1900-HB micelles exhibit much brighter FITC fluorescence at the tumor site, of which the intensity is 2.1 and 0.9 times more than that of naked micelles and G8mE1900-LB after 60 min injection. On one hand, albumin corona erected a steric barrier to inhibit the opsonin proteins adsorption and prevent macrophages uptake, thereby prolonging blood circulation time ,which could create more opportunities for MACs to accumulate in the tumor area through EPR;38 On the other hand, albumin corona


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could particularly bind to an albumin receptor (gp60) on endothelial cells and through the angiogenic tumor vasculature to increase the intratumoral accumulation.11 The difference of the biodistribution between naked micelles and MACs will decrease with time (Figure S5) because of fluorescence quenching. Consequently, albumin modification could noticeably enhance the bioavailability and biodistribution of cationic nanocarriers, reduce the accumulation of the drug in normal organs, and improve the drug concentration in the tumor site. 3.5 Antitumor Efficiency in Vitro To further investigate the treatment effects of MACs, CCK-8 assay was used against HeLa cells after 72 h. Firstly, HeLa cells cultured with medium containing MACs grow vigorously (Figure 6). Similarly, HeLa cells treated with naked G8mE1900 micelles also have cell viability higher than 90%. MACs don’t reflect the improvement of biocompatibility,39 since the naked G8mE1900 micelles already have a good cytocompatibility, with cell viability higher than 90% even at a high concentration (0.1 mg/mL).


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To test the antitumor efficacy of MACs, the cytotoxicity of PTX-loaded naked micelles and MACs toward HeLa cells after 24 h and 72 h was investigated in vitro, as well as Taxol® which is clinical-used antitumor drug as a positive control. The cell viability of G8mE1900-HB was significantly higher than that of naked micelles, while G8mE1900-LB was slightly lower than G8mE1900 under the PTX concentration in the range of 0.05 to 10 μg/ml after 24 h incubation (Figure 6C). These phenomena could be attributed to the discrepancy of cellular internalization (Supporting Information) caused by different protein corona. When the PTX concentrations were lower than 0.05 μg/ml or higher than 10 μg/ml, the cytotoxicity of all samples is in equal level within 24 h. After 72 h incubation, the growth trend of naked micelles, G8mE1900-LB and G8mE1900-HB become consistent, for all samples were internalized and thoroughly exerted encapsulated drugs after 72 h. Nonetheless, cationic micelles modified by different BSA concentration result in mutative cytotoxicity. The IC50 was calculated to be 23.07, 14.89, 38.37 and 12.27 ng/mL, respectively, for G8mE1900, G8mE1900-LB, G8mE1900-HB and Taxol®. Taxol® naturally obtained the best toxicity in vitro for it easily cross the cell membrane as a small molecule.40


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3.6 Antitumor Efficiency in Vivo Due to the complex physiological environment, nanocarriers may exhibit a completely different treatment outcome in vivo, so it is necessary and important to evaluate the therapeutic efficacy in vivo. For this purpose, naked micelles and MACs loaded PTX as key samples, drug-free naked micelles and MACs as blank controls, Taxol® as a positive control, MSA and PBS as negative controls. The samples were injected into Hela tumorbearing mice after the tumor volume reached 40 mm3. As shown in Figure 7, tumors treated with naked micelles, MACs, MSA and PBS exhibited vigorous growth. In contrast, the growth of tumors by which were treated various PTX formulations was dramatically suppressed. G8mE1900-PTX, G8mE1900-LB-PTX and G8mE1900-HB-PTX have a similar effect on tumor treatment, for the tumor growth inhibition rates are 76%, 81% and 83% compared with blank controls, and all around 80% and 84% compared with MSA and PBS, respectively. Although MACs possess abundant accumulation in tumor site as proved in distribution analysis, both naked micelles and MACs achieve similar therapy effectiveness, which may be due to the mediocre cell internalization modified by higher concentration of Albumin (Figure S3). In addition, Taxol® forcefully inhibited tumor growth


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in vivo, but its chemotherapy side effects caused decline of body weights and even two deaths at 15 and 18 days, respectively. Conversely, the body weights of mice treated with MACs and naked micelles slightly increased and all mice were alive during 21 days, suggesting that the MACs could markedly reduce the toxic side effect of the antitumor drug to normal tissues and organs. The apoptosis level and organizational damage in the tumors and the other normal tissues were also investigated by histological evaluation after treatment. As shown in Figure 7F, all PTX-loaded formulations, including G8mE1900-PTX, G8mE1900-LB-PTX, G8mE1900-HB-PTX and Taxol® display distinct apoptosis in neoplastic interior, expressed as cells shrink, nuclei lysis and membrane integrity destruction. More critically, in the case of the liver, the apoptosis level is seriously determined by the micellar formulations. The liver is the largest immune organ and serves many vital functions, and one of them is excretion foreign bodies and medications.41 As shown in Figure 7E, intravenous injection of Taxol® could result in serious hepatotoxicity of mice. The liver of mice treated with Taxol® presents large areas of necrosis, leading to the body weight loss and death of mice. Naked G8mE1900 micelles containing gemini cationic group are easily


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captured by immune cells and trapped in the liver, thereby causing certain liver toxicity. Actually, the acute hepatotoxicity of naked micelles was less than Taxol®, for the hydrophilic PEG shell protected and sustained release properties of PTX.30 Exhilaratingly, the MACs exhibited no liver damage. Once again, these results strongly confirmed that albumin modification could greatly improve the biosafety of cationic nanocarriers. As the modified albumin corona could change the physical properties and convert the charge from positive to negative on the cationic micellar surface, thereby reducing the opsonin proteins absorption in blood and the accumulation in the liver. There is no obvious toxicity to other normal tissue, including heart, lung, kidney and spleen for all formulations (Figure S6). Combined our previous research,25 we can draw a conclusion that albumin modification would not affect the excellent antitumor efficiency of gemini cationic polyurethane micelles in vivo, but also improve the biosafety and biostability. 3.7 Liver Function Tests Alanine aminotransferase (ALT) is an enzyme mostly found in liver cells. When the liver is damaged or inflamed, ALT would be released into bloodstream. Clinically, ALT concentration in the blood is commonly measured as a diagnostic evaluation on


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hepatocellular injury or liver health. To better understand the hepatotoxicity triggered by PTX-formulations, the micelles were intravenously administered into mice without xenografted Hela cells. The blank micelles without PTX, pure MSA and PBS have no harm to the liver of mice (Figure 8). All the concentration of ALT was lower than 45 U/L in the blood of mice. However, ALT concentrations treated by G8mE1900-PTX and Taxol® increased by 47% and 37% compared with PBS, indicating that both Taxol® and G8mE1900-PTX could cause liver damage. At last, the ALT concentration of G8mE900LB-PTX was increased by 22%, and the G8mE900-HB-PTX was only increased by 9%. These results demonstrate that albumin modification can indeed protect the liver from chemotherapy drugs during treatment, and improve the biosafety of the cationic nanocarriers.

4. Conclusion In summary, a new strategy to effectively improve the biodistribution and biosecurity of cationic nanoplatforms was established. Using albumin modification can change the surface physiochemical properties of cationic micelles. Protein corona with monolayer or


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multilayer was constructed depending on the different albumin concentration. Moreover, the proteins would denature as a result of the interaction with the surface of cationic micelles, and proteins in the outer layer away from the surface of the cationic micelles would keep undenatured. Multilayer Albumin without denaturation created a steric barrier to inhibit the opsonin proteins adsorption, hence prevent macrophages uptake, avoided the retention in normal tissues and enhance the drug accumulation in tumor site. MACs could significantly improve biosafety without changing the tumor therapy efficacy compared with naked cationic nanocarriers. Our work provides a promising method to modify cationic nanopaltform with optimizing the biosecurity and bioavailability for potential application in vivo.


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Figure 1. The change of size and zeta potential of naked G8mE1900 modified by various concentration BSA.


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Figure 2. TEM images of A) G8mE1900 (1 mg/ml), B) BSA protein (1 mg/ml), C) G8mE1900 with low BSA concentration (0.1 mg/ml) and D) G8mE1900 with high BSA concentration (2 mg/ml).


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Figure 3. CD spectra for BSA protein incubation with different concentration of cationic polyurethane micelles. M represents G8mE1900 micelles.


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Figure 4. CLSM images (A) and Flow cytometry (B) of Raw 264.7 cells incubated with cationic micelles modified by different BSA concentration for 0.5 h (B) and 1 h (A, B).


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Figure 5. (A) In vivo fluorescence images of tumor-bearing mice treated by fluorescentlabeled naked micelles and MACs after 60 min. (B) Quantification of the fluorescent in Liver, Kidney and Tumor after 60 min injection.


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Figure 6. Cell viability of HeLa cells after incubation with drug-free naked micelles and micelles-albumin complex for 24 (A) and 72 h (B). Cytotoxicity toward HeLa tumor cells after incubation 24 h (C) and 72 h. The IC50 values (D) of G8mE1900, G8mE1900-LB and G8mE1900-HB toward HeLa cells for 72 h of incubation.


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Figure 7. In vivo therapeutic efficacy in mice bearing HeLa tumor after treated naked micelles and MACs after 21 days. The tumor volume (A) and body weight (B), mean weights of tumors (C) and survive rates (D) changed after 21 days. H&E staining sections of liver (E) and tumors (F) separated from animals treated by different formulations. (*)


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describes statistical significance P < 0.001: the drug loaded micelles versus all the control samples. (**) P < 0.01: the drug loaded micelles versus PBS.


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Figure 8. Evaluation of alanine aminotransferase (ALT) levels after treatment with different formulations. G8 represents naked G8mE1900 micelles, G8-LB and G8-HB represent G8mE1900-LB and G8mE1900-HB, respectively. (***) P < 0.05: naked G8mE1900 micelles loaded PTX versus PBS.


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Acknowledgment This work was supported by the National Science Fund for Distinguished Young Scholars of China (51425305), the National Natural Science Foundation of China (51733005 and 51573112).

Supporting Information: The size, size distribution and zeta potential of polyurethane micelles and MAC. Drug release of naked micelles and MACs during 120 h. CLSM images and Flow cytometry of HeLa cells incubated with naked micelles and MACs for 0.5 h and 1 h. CLSM images of Raw 264.7 cells incubated for 0.5 h. In vivo fluorescence images of tumor-bearing nude mice receiving intravenous injection of fluorescent-labeled naked micelles and MACs after 6 h. H&E staining of heart, lung, kidney and spleen sections separated from animals receiving different treatments.

*Corresponding author. Fax: +86-28-85405402; Tel: +86-28-85460972; E-mail: [email protected], [email protected]


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Conflict of Interest: The authors declare no competing financial interest.


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Table of Content


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Figure 1. The change of size and zeta potential of naked G8mE1900 modified by various concentration BSA.



Figure 2. TEM images of A) G8mE1900 (1 mg/ml), B) BSA protein (1 mg/ml), C) G8mE1900 with low BSA concentration (0.1 mg/ml) and D) G8mE1900 with high BSA concentration (2 mg/ml). 148x161mm (300 x 300 DPI)


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Figure 3. CD spectra for BSA protein incubation with different concentration of cationic polyurethane micelles. M represents G8mE1900 micelles.



Figure 4. CLSM images (A) and Flow cytometry (B) of Raw 264.7 cells incubated with cationic micelles modified by different BSA concentration for 0.5 h (B) and 1 h (A, B).


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Figure 5. (A) In vivo fluorescence images of tumor-bearing mice treated by fluorescent-labeled naked micelles and MACs after 60 min. (B) Quantification of the fluorescent in Liver, Kidney and Tumor after 60 min injection.



Figure 6. Cell viability of HeLa cells after incubation with drug-free naked micelles and micelles-albumin complex for 24 (A) and 72 h (B). Cytotoxicity toward HeLa tumor cells after incubation 24 h (C) and 72 h. The IC50 values (D) of G8mE1900, G8mE1900-LB and G8mE1900-HB toward HeLa cells for 72 h of incubation.


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Figure 7. In vivo therapeutic efficacy in mice bearing HeLa tumor after treated naked micelles and MACs after 21 days. The tumor volume (A) and body weight (B), mean weights of tumors (C) and survive rates (D) changed after 21 days. H&E staining sections of liver (E) and tumors (F) separated from animals treated by different formulations. (*) describes statistical significance P < 0.001: the drug loaded micelles versus all the control samples. (**) P < 0.01: the drug loaded micelles versus PBS.



Figure 8. Evaluation of alanine aminotransferase (ALT) levels after treatment with different formulations. G8 represents naked G8mE1900 micelles, G8-LB and G8-HB represent G8mE1900-LB and G8mE1900-HB, respectively. (***) P < 0.05: naked G8mE1900 micelles loaded PTX versus PBS. 242x190mm (300 x 300 DPI)


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