Pepetide Dendron-Functionalized Mesoporous Silica Nanoparticle

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Pepetide Dendron Functionalized Mesoporous Silica Nanoparticles Based Nanohybrid: Biocompatibility and Its Potential as Imaging Probe Chunhua Guo, Jiani Hu, Leslie Kao, Dayi Pan, Kui Luo, Ning Li, and Zhongwei Gu ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.6b00093 • Publication Date (Web): 23 Mar 2016 Downloaded from http://pubs.acs.org on April 6, 2016

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ACS Biomaterials Science & Engineering

Pepetide Dendron Functionalized Mesoporous Silica Nanoparticles Based Nanohybrid: Biocompatibility and Its Potential as Imaging Probe Chunhua Guo,† Jiani Hu,§ Leslie Kao,§ Dayi Pan,† Kui Luo,*,†,‡ Ning Li,† and Zhongwei Gu*,† †

National Engineering Research Center for Biomaterials, Sichuan University, Chengdu

610064, China ‡

Huaxi MR Research Center (HMRRC), Department of Radiology, West China Hospital,

Sichuan University, Chengdu 610041, China §

Department of Radiology, Wayne State University, Detroit, MI 48201, USA

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ABSTRACT: In this study, we designed and fabricated a nanohybrid of mesoporous silica nanoparticles (MSNs) functionalized with peptide dendrons and evaluated its potential biocompatibility. The nanohybrid was prepared by combining azido-MSNs with alkynyl peptide dendrons via click chemistry to improve graft ratio. By modifying the azido-MSNs through the addition of the alkynyl peptide dendrons, we amplified and broadened the scope of their applications. After labeling with Cy5.5 dye, the nanohybrid was characterized by transmission electron microscopy (TEM), thermogravimetric analysis (TGA), nitrogen adsorption-desorption isotherms, dynamic light scattering (DLS) and zeta potential. The resulting nanohybrid showed high mono-dispersivity with a spherical diameter of 60 nm, negative surface charge and aqueous environment stability. Finally, a systematic assessment was conducted to evaluate the biocompatibility of the MSN-dendron-Cy5.5 based nanohybrid, both in vitro and in vivo. Cytotoxicity measurements, body weight shifts, histological analysis and routine blood tests with mice demonstrated that the nanohybrid had good biocompatibility. Hemocompatibility evaluations demonstrated that the nanohybrid had improved blood safety compared to bare MSNs. Healthy nude mice were used to analyze the in vivo optical fluorescence images. Ex vivo fluorescence images of the major organs were studied to further evaluate the in vivo biodistribution of the nanohybrid, with results suggesting that the MSN-dendron-Cy5.5 based nanohybrid provided promise in biomedical applications. Overall, we provided a preliminary but important research piece on the safety and 2 ACS Paragon Plus Environment

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efficiency of MSN-dendron-Cy5.5 based nanohybrid as an in vivo functional vehicle used in diagnosis and therapy. KEYWORDS: mesoporous silica nanoparticles, peptide dendrons, nanohybrid, biocompatibility, near-infrared fluorescence imaging 1. INTRODUCTION With the maturation of nanotechnology, interest has grown surrounding the use of nanomaterials in disease therapy and diagnosis.1,2 Among the different types of nanomaterials, mesoporous silica nanoparticles (MSNs) have demonstrated promise as an alternative vehicle for imaging probes and drug delivery in the biomedical field.3-5 The unique features of MSNs, such as high surface area, customizable pore diameter, large pore volume, easy surface modification and physicochemical stability have proved to be beneficial to tailor MSNs into medical imaging applications.6,7 However, applications of unfunctionalized MSNs for loading drug molecules via non-covalent interactions were limited by their poor pharmacokinetics and low efficiency.8,9 Previous studies also have suggested that unfunctionalized MSNs could induce enhanced hemolytic activity, which may be ameliorated via surface modification.10,11 With the increasing attention given to multifunctional and safe nanocarriers for biomedical application, MSNs have been modified with different kinds of moieties and groups, such as dendrimers, proteins, antibodies, iron oxide core, etc.12-15 The 3 ACS Paragon Plus Environment


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functionalization of MSNs may therefore increase the versatility and broaden the horizons of MSNs’ applications.16-18 Some studies have reported that PEG-functionalized MSNs have a long half-life in systemic circulation and a high biocompatibility.19,20 Dendrimers/dendrons that are defect free with complete monodispersivity, high end-group functionality, compact and precisely defined molecular structures have been employed to modify MSNs and extend their biomedical application.12, 21 However, until now, most of dendrimers utilized in MSNs modification were PAMAM dendrimers, which were not stable in solution and may be cytotoxic, secondary to their nondegradable and hemolytic effects.22-24 Compared with PAMAM and other dendrimers, the peptide dendrimers and dendrons show a better biocompatibility and immunocompatibility and have been widely used as new-generation biomaterials.25 Previous reports demonstrated that the half-life of peptide dendrimers/dendrons in systemic circulation was size dependent and directly proportional to its molecular weight.26 However, difficulty has arisen in balancing the long half-life of high-generation dendrimers/dendrons with increasing side effects.27 Recently, MSNs have been employed as imaging probes for near-infrared fluorescence (NIRF) imaging.28,29 Unfortunately, there are some challenges in NIRF imaging sensitivity. NIRF in vivo imaging can be limited by light and infrared absorption through the interference of water and hemoglobin present in biological tissues.30 NIRF image sensitivity thus depends on proper filtering and selective passage of fluorescent light to 4 ACS Paragon Plus Environment

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the NIRF detector. Sensitivity is also affected by background interference light leakage. While increased amounts of imaging agent can increase sensitivity of image generation, concomitantly increase side effects in investigational human studies.31,32 In vivo imaging with the MSN-dendron nanohybrid can bypass these crucial limitations because the NIRF excitation and emission light are able to pass through the optically transparent mesoporous silica matrix. 28 With the attachment of the organic dye Cy5.5, a synthetic fluorophore used as an optical imaging agent, which has a fluorescence excitation and emission wavelengths of 673 nm and 692 nm, respectively, thus the nanohybrid can also minimize intrinsic background interference by bypassing the absorbance spectrum of blood, lipids and other tissues that are in the range of NIRF wavelengths .28, 33, 34 Based on the above observations, in order to overcome the shortcomings of using MSNs and low molecular weight dendrimer/dendrons separately, we combined both materials to fabricate a nanohybrid through the synthesis of the MSNs with peptide dendrons and NIRF dye Cy5.5. The resulting fabricated hybrid nanoparticle had a spherical diameter of 60 nm, which fell within the 50–100 nm range considered acceptable for potential drug and gene delivery vehicles and imaging probes.1 Subsequently, we studied the efficacy and biocompatibility of our fabricated MSN-dendron-Cy5.5 based nanohybrid and analyzed possible applications in NIRF optical imaging. 2. EXPERIMENT SECTION  5 ACS Paragon Plus Environment


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2.1.

Materials

and

Measurements.

Trifluoroacetic

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acid

(TFA),

N,N-diisopropylethylamine (DIPEA), succinic anhydride, and Mono-NHS ester Cy5.5 were purchased from Sigma-Aldrich and used without further purification. The morphology of MSNs and the organic-inorganic nanohybrid materials were elucidated on a JEM-100CX (JEOL) transmission electron microscope (TEM) by dropping the samples onto a carbon-coated copper grid with a concentration of 100 μg/mL. Dynamic Light Scattering (DLS) and zeta potentials were measured by Zetasizer Nano ZS (Malvern Instruments, Worcestershire, UK) with a final concentration of 100 μg/mL in the phosphate buffered saline (PBS) buffer. Fourier transform infrared spectra (FTIR, PE spectrometer) were collected in the 400-4000 cm-1 range. The properties of the mesoporous structure were determined by nitrogen adsorption-desorption experiments on a Micromeritics Gemini VII 2390 Surface Area Analyzer. Samples were degassed for 12 h at 120 oC under nitrogen prior to measurements. The organic moieties were detected by thermogravimetric/differential thermal analysis (TG/DTA) on a NETZSCH-Leading Thermal Analysis in the temperature range of 35-1000 oC. 2.2. Synthesis of Mesoporous Silica based Peptide Dendritic Nanohybrid. The MSNs were synthesized according to the method delineated in previous literature.35 The detailed method of the synthesis of dendronized-MSNs has been previously described in our past reports.36 In a nitrogenous atmosphere, dendronized-MSNs (100 mg) were dispersed in an anhydrous solvent of dichloromethane (CH2Cl2, 3 mL) and trifluoroacetic 6 ACS Paragon Plus Environment

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(TFA, 3 mL). The subsequent mixture was placed into an ice bath and the mixture was stirred for 2 h at room temperature. After the solvent was removed by rotary evaporation, anhydrous diethyl ether was added and a precipitate appeared. The precipitate was then centrifuged and washed 3 times with anhydrous diethyl ether. The unprotected product was treated with DIPEA (0.5 mL) in anhydrous DMSO under a nitrogenous atmosphere. Mono-NHS ester Cy5.5 (0.5 mg) diluted in DMSO was added and the mixture stirred under nitrogen in an ice bath for 30 min and then at room temperature for another 24 h, away from light. After several rounds of centrifugation and washing with ethanol and water, the mixture was further purified via water dialysis and collected by freeze-drying. Once collected, the dendronized MSNs labeled with Cy5.5 (80 mg) was mixed in dimethylformamide (DMF) and treated with DIPEA (0.1 mL), followed by the addition of succinic anhydride (24 mg). The succinic anhydride modified the amino groups on the dendronized-MSNs that did not completely react with mono-NHS ester Cy5.5, improving the hydrophilicity and dispersal of the hybrid in water. The samples were then centrifuged and washed 3 times with DMF and water, and further purified via water dialysis. The final product (Scheme 1. MSN-dendron-Cy5.5 based nanohybrid) was redispersed in water and stored at 4 oC before being used. 2.3. Blood Compatibility Studies. Blood was collected from healthy volunteers taking no medications and stored in sodium citrate tubes with a blood/anticoagulant ratio of 9:1.



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The blood compatibility evaluations were operated according to the accepted parameters and previous reports.37-39 2.3.1. Prothrombin Time (PT) and Activated Partial Thromboplastin Time (APTT). Platelet-poor plasma was prepared by centrifuging the citrated whole blood at 3000 g for 15 min at room temperature. The supernatant plasma (360 μL) was then mixed with 40 μL of MSNs or nanohybrid dispersion in the PBS buffer for final concentrations of 0.1 mg/mL, 0.5 mg/mL and 1 mg/mL. The same volume of PBS solution was used as a control. Measurements were performed at 37 °C on a STA-R Evolution automatic coagulation analyzer (Stago Company, France) with the coagulation reagents added. Each experiment was repeated separately three times. 2.3.2 Thromboelastography (TEG). Fresh whole blood (900 μL) was mixed with 100 μL PBS dispersion of MSNs or nanohybrids in a tube containing kaolin for a final concentration of 0.1 mg/mL and 1 mg/mL of materials in blood. Pure PBS solution was used as a control. After thorough mixing, 340 μL of the mixed dispersion was added to a TEG cup for coagulation analysis at 37 °C. TEG analysis was initiated by adding 20 μL of 0.2 M CaCl2 solution to each sample in the cup and tested by the TEG analyzer.  2.3.3. Red Blood Cell Morphologies and Aggregation. A red blood cell (RBC) suspension was prepared by centrifuging the citrated whole blood at 1000 g for 5 min at room temperature. After the plasma and buffy coat layers were removed, the RBCs were washed with PBS for 3 times. Then 20 μL RBCs were mixed with 100 μL of MSNs or 8 ACS Paragon Plus Environment

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nanohybrid dispersion in the PBS buffer for a final concentration of 0.1 mg/mL, 0.5 mg/mL and 1 mg/mL after vortexing. A PBS buffer solution with an equal volume was used as a control. The mixture was incubated for 15 min, then washed with PBS and fixed with 4% paraformaldehyde overnight. The suspensions were dropped on glass slides, dehydrated with 75, 85, 95, 100% (v/v) ethanol for 10 min respectively and then dried overnight in air at room temperature. The dried RBCs samples were coated with gold and further analyzed used scanning electron micrography (SEM) 2.3.4. Red Blood Cell Hemolysis Assay. 50 μL diluted RBC suspensions were added to 1 mL PBS dispersions of MSNs or nanohybrids for final concentrations of 0.1 mg/mL, 0.5 mg/mL and 1 mg/mL. The samples of erythrocytes incubated with deionized water and PBS were used as the positive and negative control, respectively. All the samples were kept in static condition at room temperature for 12 h then centrifuged at 1000 g for 5 min. 200 μL of supernatant of each samples were transferred to a 96well plate. A microplate reader with a reference absorbance of 655 nm was used to determine the absorbance values of the supernatant at 540 nm. Finally, the erythrocyte hemolysis percentage was calculated using the following formula: Hemolysis percentage (%) = [(sample absorbance - negative control absorbance) / (positive control absorbance negative control absorbance)] × 100. The hemolysis percentage for each sample was repeated three times. 



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2.4. In Vitro Cytotoxicity Test. The in vitro biocompatibility of the nanohybrid and bare MSNs was measured by a Kit-8 assay (CCK-8, Dojindo, Japan). Four cell lines, human embryonic kidney cell lines (293T), human liver cell lines (L02), human cervical cancer cell lines (Hela) and mouse myoblast cell lines (C2C12) were cultured in DMEM medium with 100 IU/mL penicillin, 100 μg/mL streptomycin and 10% fetal bovine serum at 37 oC in a humidified atmosphere of 5% CO2. The cells were then digested by Trypsin-EDTA (GIBCO) and seeded in a 96-well plate at a density of 5000 cells per well before being cultured in 5% CO2 at 37 oC for 24 h. The nanohybrids and the non-functionalized MSNs with the same concentrations (10, 25, 50 and 75 μg/mL) in DMEM medium were added to each of the wells. The cells were further incubated for 24 h for measurement. Cell viability of the untreated control cells was set as 100%. 2.5. In Vivo Toxicity. Animal experiments were conducted in accordance to local and national welfare legislation. Normal female BALB/c mice, 6-8 weeks old (20 ± 2 g, n = 5 for each studied group) were used to carry out the experiments. The control group was given saline and the two experimental groups were injected with either MSNs or nanohybrid, at a dose of 20 mg/kg via the tail vein every 6 days for 21 days. The mice’s body weights were measured and their behavior was monitored. One week following the final injection, all three groups of mice were sacrificed. Blood was collected for the routine blood tests and the organs of interest (liver, lung, heart, spleen and kidney) were separated, washed with PBS and fixed in 4% formaldehyde for histological analysis.  10 ACS Paragon Plus Environment

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2.6. In Vivo and Ex Vivo Imaging Studies. In vivo fluorescent imaging was performed using a Maestro in vivo imaging system (Cri, USA). Healthy female nude mice were fasted 12 h prior to imaging. A 20 mg/kg dose of the MSN-dendron-Cy5.5 nanohybrid was injected into the tail vein. Optical images of the whole body were taken at 0 h, 1 h, 6 h, 24 h, 54 h and 94 h after injection of the MSN-dendron-Cy5.5 nanohybrid. In order to estimate the biodistribution of the optical imaging probe, the mice were sacrificed at 6 h and 24 h after the nanohybrid administration. Liver, spleen, heart, lung, and kidney were then dissected and imaged ex vivo using the same in vivo imaging system.

3. RESULTS AND DISCUSSION  3.1. Synthesis and Characterizations of MSN-dendron-Cy5.5 Based Fluorescence Imaging Nanohybrid. In this study, we designed a nanohybrid fluorescent imaging nanoparticle by conjugating peptide dendrons to mesoporous silica nanoparticles, as shown in Scheme 1. A challenge we encountered in grafting the peptide dendrons onto MSNs was steric hindrance stemming from the specific geometry of the two compounds. In order to improve the reaction’s graft ratio, the peptide dendron was functionalized with alkynyl groups, while the MSNs’ surface was functionalized with azido groups. Cu(I) catalyzed the azide-alkyne cycloaddition was assisted in grafting the alkynyl terminated peptide dendrons to the azido-functionalized MSNs. As shown in Figure 1, the peak of 11 ACS Paragon Plus Environment


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2126 cm-1 assigned to the IR absorption of the azido group in the IR spectra of MSNs-N3 disappeared in that of the MSN-dendron-Cy5.5, indicating the success of “click” reaction. In addition, the peak at 1256 cm-1 attributed to the IR absorption of sulfonic groups in Cy5.5, further confirmed the successful attachment of Cy5.5 onto the surface of MSN-dendron. Both the morphology and size of the MSNs and nanohybrid were observed by TEM. As shown in Figure 1A, non-functionalized MSNs had a spherical shape and an average particle diameter of about 60 nm. The porosity of the MSNs could be clearly seen in Figure 1A at this magnification. Figure 1B shows the MSN-dendron-Cy5.5 based nanohybrid. Due to the small size (thickness) of the peptide dendrons, no significant size difference was observed. The morphology of the functionalized nanohybrid can also be visualized despite the presence of the thin grafted peptide dendron-Cy5.5 layer. DLS was used to analyze the size and size distribution of the nanoparticles. The average hydrodynamic diameter of the non-functionalized MSNs, as determined by DLS, was 83.2 ± 3.1 nm (PDI = 0.096 ± 0.009) (Figure 2C and Table 1) versus 60 nm as determined by TEM imaging. The MSNdendronCy5.5 nanohybrid’s hydrodynamic diameter was measured at 106.4 ± 2.1 nm (PDI = 0.248 ± 0.024) (Figure 2D and Table 1), which was again larger than that determined by TEM. These size differentials depending on measuring platform could most likely be attributed to the environmental state with which the tests were taken. The TEM measurements were taken in the sample’s dry state, 12 ACS Paragon Plus Environment

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while the DLS measurements reflected the hydrodynamic diameter of samples, which was influenced by both particle-particle interactions (electrostatic, hydrodynamic, etc) and peptide dendrons layer swelling around the particles. The zeta potential of the MSNs and the nanohybrid were -14.0 ± 0.98 mV and -17.2 ± 0.46 mV, respectively (Table 1). The slight increase in the zeta potential of the MSN-dendron-Cy5.5 might be attributed to the sulfonic groups of Cy5.5 and the carboxyl groups created through the succinic anhydride modification of the MSN-dendron nanohybrid. Materials with negative zeta potential had no side effects on cells, organs and other physiological environments, thus representing the safety of the nanohybrid in vivo. Additionally, the nitrogen absorption-desorption isotherm (Figure 3) displayed a type-IV isotherm, which was the characteristic of the mesoporous materials.40 Finally, the surface area of the nanohybrid (24.6 m2/g) was significantly smaller compared to the non-functionalized MSNs (231.4 m2/g) due to the successful modification of MSNs with the peptide dendron moieties. Collectively, these above results demonstrated the successful synthesis of the MSN-dendron-Cy5.5, creating a nanohybrid with a diameter of 60 nm, surface area of 24.6 m2/g and negative zeta potential.  TGA measurements were employed to quantify the peptide dendrons and other organic ligands conjugated onto the surface of MSNs (Figure 4). For unfunctionalized MSNs, the weight loss was only observed at one reaction stage within a temperature range of 35 oC to 120 oC, which could be attributed to the loss of physically absorbed water (3%, weight 13 ACS Paragon Plus Environment


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percent). For the functionalized dendron nanohybrids, the decomposition process was divided into three stages within a temperature range of 35 oC to 1000 oC. The first stage, from 35 oC to 120 oC, had a corresponding weight decrease less than 3%, which can be attributed to the evaporation of physically adsorbed water similar to the nonfunctionalized MSNs’ response. The second weight loss stage was seen between 150 o

C to 420 oC and attributed to the decomposition of the peptide dendrons and Cy5.5

ligands that were previously immobilized on the MSNs surfaces. Finally, the TGA curves dropped in the third temperature range, 420 oC to 680 oC due to the breaking of the covalent bonds between the MSNs and dendrons, consistent with our previously reported results of nanoparticles functionalized with peptide dendrimers.41 Using these TGA results, peptide dendrons and other organic ligands covering the MSNs contributed to 27% of the total functionalized MSNs weight content. 3.2. Hemocompatibility. The hemocompatibility of the MSNs and nanohybrid were evaluated by measuring blood coagulation, erythrocyte morphologies, aggregation and lysis under in vitro conditions. Plasma coagulation properties were used as a barometer for interaction between biomaterials with blood components.42 Prothrombin time (PT) and activated partial thromboplastin time (APTT) were measured to determine the pro-or anti-coagulant nature of MSNs and nanohybrid at various dispersion concentrations (0.1 and 1 mg/mL) in human plasma. Controls were created by adding identical volumes of PBS to blood sample. Theoretically, PT reflects the extrinsic and common coagulation 14 ACS Paragon Plus Environment

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pathway and experimentally indicates the time to the formation of a fibrin clot after tissue thromboplastin is added. APPT characterizes the intrinsic and common coagulation pathways and experimentally represents the formation time of a fibrin clot in the plasma after a partial thromboplastin reagent and calcium chloride have been added.39 The effects of MSNs and nanohybrid on PT and APTT were shown in Figure 5. The results showed that all PT and APTT data fell within the normal range (the area between the two dotted lines). However, Figure 5A revealed that the MSNs’ APTT was statistically elevated (*p < 0.05) compared to that of the PBS control, reflecting the fact that MSNs may precipitate side effects in the intrinsic and common coagulation pathways. In contrast, the nanohybrid did not produce any significant impacts on the intrinsic, extrinsic or common coagulation pathways when compared to the PBS buffer control. Compared to APTT and PT studies, thrombelastograph (TEG) parameters can replicate in vivo conditions more accurately and can determine the effects on coagulation properties when whole blood comes into contact with the biomaterials.43 Using TEG we analyzed the formation and strength properties of a blood clot as a function of time. Four principle parameters related to the kinetics and strength of clot formation were investigated: R, the time from the start of a run until the initial signs of a detectable fibrin formation; K, the period from the start of the test to substantial clot formation, representing the dynamics of clot formation; α angle, similar to K, reflecting the rate of clot polymerization or rate of fibrin cross-linking to characterize clotting time; and MA 15 ACS Paragon Plus Environment


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(maximum amplitude), an analysis of maximum clot strength or stiffness. With these values, TEG can characterize the overall process of whole blood coagulation and provide a better clinical and experimental picture of in vivo or in vitro coagulation.44 The TEG traces of clot formation in whole blood in the presence of MSNs and nanohybrid with a respective concentration of 0.1 and 1 mg/ml were given in Figure 6, while the main parameters summarized from the TEG graphs were shown in Table 2. Analyzing these results, MSNs with different concentrations led to abnormal TEG values. R values were decreased indicating that MSNs shortened the time to initial fibrin formation. α values were increased, suggesting MSNs accelerated the rate of fibrin formation. More plainly, from the results of the TEG study, it appeared that MSNs had a procoagulant effect on the clotting process. Analyzing the nanohybrid TEG results, the R values were in normal range. Meanwhile, the other principle parameters also fell within the normal rage and did not impact coagulation due to the addition of the nanohybrid when compared with the PBS control. The observed decrease in R values may be due to the larger MSNs surface area, which caused an undesirable interaction with the coagulation factors and led to the premature activation of coagulation system. Similar reasoning may have caused the observed increase in α values. Thus, MSNs induced a hypercoagulable state, increasing the risk of a thrombus formation. However, once modified with the peptide dendrons, the nanohybrid reverted to a smaller surface area


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with a more negative charge. Compared to the uncoated MSNs, the nanohybrid had very little effects on the overall blood clotting process as observed in the TEG data. Due to their high count and volume fraction in blood, red blood cells (RBCs) are widely used to evaluate the impact of biomaterials on blood in biocompatibility analysis. Once biomaterials are administered intravenously, the interaction between the foreign substance and intravascular erythrocytes may precipitate potential side effects.45 The erythrocyte membrane is a practical predictive model that analyzes the interactions between mammalian cell membranes and foreign materials. Erythrocyte morphology, aggregation and hemolysis are important parameters in assessing the hemocompatibility of biomaterials. The effects of MSNs and nanohybrid on erythrocyte morphology and aggregation were analyzed by SEM. As displayed in Figure 7, the erythrocytes were incubated with various concentrations of MSNs or nanohybrid resulting in adequate dispersal and normal biconcave morphology as compared to the PBS control group. The smooth surface of the erythrocytes membranes signified that the cell membrane was intact. Accordingly, both the MSNs and nanohybrid displayed a safe profile towards the RBCs with no significant effects on their morphologies or aggregation. Figure 8 illustrated the hemolysis assay. Uncoated MSNs caused severe hemoglobin release from damaged RBCs. The uncoated MSNs affected the percent hemolysis in a directly concentration-dependent way. MSNs with concentrations of 0.1 mg/mL, 0.5 mg/mL and 1 mg/mL induced 43%, 64% and 68% lysis of RBCs, respectively. The high 17 ACS Paragon Plus Environment


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hemolytic activity of uncoated MSNs far exceeded the acceptable hemolysis percentage threshold of 5% and may be attributable to the bare MSNs’ larger surface area per mass. This increased proportion resulted in a higher number of exposed silanol groups on the contact surface between the erythrocytes and MSNs. Similar results were observed in previously published studies.10 In contrast to the MSNs, no apparent hemolytic activity was observed after RBCs incubation with hybrid nanoparticles over 12 h. The MSN-dendron-Cy5.5 based nanohybrid ameliorated the hemolytic activity due to the less exposed silanol groups on the contact surface with decreased surface area compared to the bare MSNs and the improved biosafety of the peptide dendrons. This simple surface modification with bio-safe groups was a promising solution to ensure the safe use of MSNs in clinical applications or laboratory research. 3.3. The Cytotoxicity and In Vivo Toxicity Test. The in vitro cytotoxicity of the MSN-dendron-Cy5.5 based nanohybrid was evaluated by CCK-8 assays on 293T, L02, Hela and C2C12 cell lines after a 24 h incubation period, then compared with that of uncoated MSNs. As shown in Figure 9, the cell viabilities of the nanohybrid and the uncoated MSNs in each cell line were greater than 80% for each measured concentration (from 10 μg/mL to 75 μg/mL), suggesting the materials had no significant toxicity on the experimental cells. The low in vitro cytotoxicity of the nanohybrid should be due to the excellent biocompatibility profile of MSNs and peptide dendrons (less than five generations).46 18 ACS Paragon Plus Environment

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For any biomaterials, in vivo toxicity was an important criterion to evaluate before further utilization in real applications.47 Evaluations were based on tracking body weight shifts and observing changes in the mice's behavior. To systematically assess the risk profile of the nanoparticles, healthy BALB/c mice were administrated with nanohybrid, MSNs and physiological saline as a control. The mice were injected with a 20 mg/kg dose of their respective agent once every 6 days for 21 days total. Throughout the evaluation period, no obvious signs of dehydration, locomotor impairment, muscle loss, anorexia or other focal or systemic symptoms associated with animal toxicity were observed. Figure 10 showed the body weight shifts of the healthy mice over the 21-day experimental period. Both groups of mice administrated with either MSNs or nanohybrid showed similar body weight shifts to that of the physiological saline group. No abnormal body weight loss or mortality was observed. These results suggested that the nanohybrid did not induce significant nutritional or metabolic toxicity. To further investigate potential toxicities of the MSN-dendron-Cy5.5 based nanohybrid on healthy mice, a routine blood test and histological analysis were performed on day 21. Representative hematology markers were measured, including plateletcrit (PCT), platelet count (PLT), red blood cell count (RBC count), mean platelet volume (MPV), hemoglobin (HGB), mean corpuscular hemoglobin (MCH), white blood cell count (WBC count), hematocrit (HCT) and mean corpuscular volume (MCV). As shown in Figure 11, the measured parameters of each experimental group were found to be normal as compared to that of 19 ACS Paragon Plus Environment


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the control group. The results suggested that the nanohybrid could be considered safe with no observable side effects on blood components. In contrast to the above hemocompatibility analysis, an additional Routine Blood Test was taken from a living mouse. Since the mouse’s in vivo immune system might protect the blood products from side effects, a hemocompatibility analysis in vitro was conducted to reveal any potential side effects of materials on the organism. A series of biocompatibility evaluations were then necessary to complete the risk profile of biomaterials.48 Histological analysis was performed to explore whether the nanohybrid or its degradation products could cause tissue damage, inflammation or lesions secondary to toxic exposure. As shown in Figure 12, five organs from mice injected with MSNs and MSN-dendron-Cy5.5 based nanohybrid all had pathologically normal structures with no obvious abnormalities compared to the control group. No degeneration was observed in the cardiac myocyte samples. Liver samples showed normal hepatocytes with no inflammatory infiltrates. Neither hyperplasia nor pulmonary fibrosis was found in the spleen or lung samples respectively. The renal glomerulus was intact and easily visible in the kidney samples. These results corroborated the lack of any pathological abnormalities occurring in any of the experimental animals. The complete toxicity profile of the nanoparticles depended on their size, shape, structure, charge, chemical composition, metabolism, surface functionality, molecular weight

and

degradability

characteristics.49

The

high

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MSN-dendron-Cy5.5 based nanohybrid may be attributed to their compact globular morphology and negative surface charge that maintains the nanoparticles’ stability in vivo. Additionally, the biodegradability of both the MSNs and peptide dendrons could promote their clearance from the organism, enhancing their in vivo biocompatibility.46, 50,51 Not only did we analyze the nanohybrid's effects on cell lines and blood components in vitro, but also in live mouse experiments and did a further side-by-side analysis of the nanohybrid versus the uncoated MSNs. Our analysis showed that the nanohybrid had no significant toxicity to cells, blood and healthy organs and had improved biosafety compared to the uncoated MSNs. Therefore based on our above analysis, MSN-dendron-Cy5.5 based nanohybrid produced no obvious toxicity and could be considered as a potential nanoscale optical imaging probe. 3.4. In Vivo and Ex Vivo Imaging Studies. As one modality in the burgeoning optical imaging field, NIRF, like all types of optical imaging reduces patients’ exposure to harmful radiation, compared to the current nuclear imaging techniques. NIRF has been applied to fields such as cancer imaging and minimally invasive surgery, allowing for intraoperative, real-time visualization and identification of anatomy.32, 52-54 Finally, healthy BALB/c nude mice were used to evaluate the possibility of using MSNs-dendron-Cy5.5 based nanohybrid as a contrast agent for NIRF imaging at different time points. As shown in Figure 13A, 1 h after intravenous injection of the nanohybrid, strong NIRF signals were observed at the hepatic site, while the signals in other parts of 21 ACS Paragon Plus Environment


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the body were not grossly visible. At 6 h, 24 h, 54 h and 94 h post-injection, the hepatic site signals were still strong with a gradually decreasing trend over time. At 94 h post-injection, the image reflected the hepatobiliary excretion progress of the nanohybrid materials.51 The whole body images suggested that the MSN-dendron-Cy5.5 based nanohybrid could accumulate in the liver post-injection. Thus the MSN-dendron hybrid could be used as a vehicle that would help extend circulation time within a safe environment for the NIRF contrast agent when injected in the body. Oftentimes, the deep-positioned tissues or organs in mice are hard to visualize in vivo by fluorescent methods due to limited light penetration, which in turn affects fluorescence signal intensity.32, 55 To accurately acquire a quantitative biodistribution of the MSN-dendron-Cy5.5 based nanohybrid in different organs in vivo, ex vivo imaging studies were carried out at 6 h and 24 h after intravenous injection of the nanohybrid. As shown in Figure 13B, fluorescence imaging of major organs: the liver, lung and spleen registered a strong fluorescence signal 6 h post-injection of the MSN-dendron-Cy5.5 based nanohybrid. The fluorescent intensity of different organs (Figure 13C) quantitatively confirmed the highest accumulation of the MSN-dendron-Cy5.5 based nanohybrid in lungs, followed by the liver with hardly any accumulation in the other organs at 6 h. However, ex vivo imaging of organs at 24 h indicated that the fluorescent intensity in lungs decreased significantly, while the liver still exhibited high-intensity fluorescence. In vivo imaging of the lung showed no signal, which might be due to its 22 ACS Paragon Plus Environment

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position in the body (too deep for light penetration). With regards to the liver, the fluorescence signal was easily observed because of its large size and low position in body. In vivo and ex vivo imaging studies suggested that the MSN-dendron-Cy5.5 based nanohybrid primarily targeted the lungs and liver post-injection over a short period of time. The liver, unlike the lungs, was able to emit a strong fluorescence signal for at least 4 days post-injection in our studies. Taking advantage of our nanohybrid’s affinity for both liver and lung tissue, its high biocompatibility profile and long half-life, several different therapeutic and diagnostic applications emerged. Specifically, NIRF image guided surgery has recently been used to enhance current lung cancer treatments through increasing treatment modalities and hopefully survival rates for the second leading cause of cancer-related deaths.53,54 Despite pre-surgical radiologic imaging, nodules

Pepetide Dendron-Functionalized Mesoporous Silica Nanoparticle

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