Magnetic Hybrid Nanoparticles with Folate-Conjugated

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Luminescent/Magnetic Hybrid Nanoparticles with Folate-Conjugated Peptide Composites for Tumor-Targeted Drug Delivery Jian-Min Shen,†,‡ Xing-Mei Guan,† Xiao-Yan Liu,† Jing-Feng Lan,† Ting Cheng,† and Hai-Xia Zhang*,† †

Key Laboratory of Nonferrous Metal Chemistry and Resources Utilization of Gansu Province and ‡School of Life Sciences, Lanzhou University, Lanzhou 730000, China S Supporting Information *

ABSTRACT: We developed a novel chitosan-based luminescent/magnetic hybrid nanoparticles with folate-conjugated tetrapeptide composites (CLMNPs-tetrapeptide-FA) by conjugation in situ. First, chitosan, CdTe quantum dots (QDs), and superparamagnetic iron oxide were directly gelled into ternary hybrid nanogels. Subsequently, tetrapeptides (GFFG and LGPV) and folate were conjugated orderly into the hybrid nanoparticles. The morphology, composition, and properties of the as-prepared copolymers have also been characterized and determined using TEM, EDX, XRD, FTIR spectra, DLS, fluorescence spectroscopy, VSM, and fluorescence microscopy imaging studies. The size range of the end product CLMNPs-tetrapeptide-FA copolymers was from 150 to 190 nm under simulated physiological environment. In vivo, the experimental results of magnetic accumulation showed that the copolymers could be trapped in the tumor tissue under magnetic guidance. Under the present experimental conditions, the loading efficiencies of CPT were approximately 8.6 wt % for CLMNPs-GFFG-FA and 1.1 wt % for CLMNPs-LGPV-FA, respectively. The CPT cumulative release under dialysis condition mainly occurred for the first 28 h, and could reach 55% at pH 5.3 and 46% at pH 7.4 from CPT-loaded CLMNPs-GFFG-FA, and 69% at pH 5.3 and 57% at pH 7.4 from CPT-loaded CLMNPs-LGPV-FA within 28 h, respectively. The hemolysis percentages (98%) by HPLC. The ESI-mass spectrometry of the resulting peptides was performed on Bruker maXis 4G spectrometer equipped with a nebulizer-assisted electrospray source (see SI 1). Then, 53.7 μmol of GFFG-NH-Fmoc and LGPV-NH-Boc were dissolved in 7 mL of anhydrous DMSO and 7 mL of methanol, respectively. After shaking gently for 5 min, the mixtures were kept at room temperature overnight. The absorbances of the GFFG-NH-Fmoc and the LGPV-NH-Boc solution were recorded from 250 to 450 nm (see SI 2). Afterward, to conjugate the tetrapeptide linkers with chitosan on the CLMNPs surface by amido bond, the end carboxyl groups on two tetrapeptides were activated by adding 30 mg of NHS and 53 mg of EDAC and stirring at room temperature for 2 h. Finally, the previous resulting CLMNPs were divided equally into two parts and added to the two mixtures, respectively. After shaking on a level swing bed at room temperature for 12 h, the reaction mixtures were centrifuged at 10 000 rpm for 5 min. The precipitate containing GFFG was washed three times with DMSO, and the precipitate containing LGPV was washed three times with methanol. The products were denoted as CLMNPs-GFFG-Fmoc and CLMNPs-LGPV-Boc. All supernatants from the two systems were collected, respectively, and the absorbance values were measured at 296 nm for free GFFG-Fmoc and at 374 nm for free LGPV-Boc. The loading amounts of two tetrapeptides on CLMNPs were calculated by deducting free tetrapeptide content from added total amount. To obtain the final compound CLMNPs-GFFG-NH2 and CLMNPs-LGPV-NH 2 , the deprotection procedures of CLMNPs-GFFG-Fmoc and CLMNPs-LGPV-Boc were indispensable. In the case of CLMNPs-GFFG-Fmoc, 3 mL of 5% piperidine solution (in anhydrous dimethyl formamide, DMF) was added. After shaking gently for 5 min at room temperature, the mixture was centrifuged at 10 000 rpm for 5 min. The precipitate was treated again with 3 mL of 5% piperidine solution by shaking for 20 min. Afterward, the precipitate was washed three times with cold anhydrous DMF in order to obtain purified CLMNPs-GFFG-NH2. With respect to the deprotection of CLMNPs-LGPV-Boc, 10 mL of trifluoroacetic 1012

dx.doi.org/10.1021/bc300008k | Bioconjugate Chem. 2012, 23, 1010−1021

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at 366 nm. The loading efficacy and loading amount of CPT were determined according to the decrease in initial CPT solution. All the experiments were carried out in triplicate. In order to evaluate the effect of pH on the release mechanism, the release profiles of CPT were obtained by the dialysis method in a simulated normal body fluid (PBS, pH 7.4) and an acidic environment (PBS, pH 5.3) at 37 ± 1 °C. First, 30 mg of the CPT-loaded CLMNPs-GFFG-FA and CLMNPsLGPV-FA copolymers were dispersed in 5 mL of medium and placed in a dialysis bag (molecular weight cutoff of 14 kDa), respectively. The dialysis bags were then soaked in 45 mL of the release medium in a water bath with gentle shaking. 3.0 mL aliquots of sample in the solution were withdrawn at defined time periods, and as soon as the amount of CPT released was analyzed by UV spectrophotometry at 366 nm, the measuring solution was returned to the medium. Each experiment was conducted in triplicate and results are presented as mean (standard deviation). Magnetic Targeting in Vivo. In order to assess magnetic targeting of copolymers in vivo, S-180 sarcoma-bearing Kunming mice (male, body weight 18−20 g) were used. After mice were anesthetized by intraperitoneal injection of 20% chloral hydrate, 0.2 mL of the blank CLNMPs-GFFG-FA copolymer with a concentration of 200 μg/mL in sterile PBS were injected by tail vein. Subsequently, mice were fastened and treated with a constant magnet field (about 1500 Oe) laid outside the skin of the belly near the tumor site at 25 °C (Figure 4C). After three hours, the mice were killed, and the tumor tissues were quickly removed and cut into slices (thickness about 20 μm) on a freezing microtome (Leica CM1100, Germany). The magnetic copolymer accumulation in the tumor slice was detected on a fluorescence microscope (Nikon TE2000-S, Japan) by observing luminescence imaging under excitation wavelength of 488 nm. The statistics of the magnetic copolymer trapped in the tumor tissue or cells were measured via photoluminescence (PL) intensity in a microscopic field. The control experiment was also performed (other operations similar except no magnet field applied). Each experiment was conducted in triplicate and results are presented as mean (standard deviation). Haemacompatibility Assessment in Vitro. Hemolysis Assay in Vitro. Three mL of human blood was centrifuged at 1500 rpm for 10 min to acquire human red blood cells (HRBCs). After removing the serum by suction, HRBCs were washed five times with sterile physiological saline solution by centrifugation and suction, and diluted to 10-fold volume to make the absorbance value of the positive control supernatant located in the range 0.5−0.6 at 541 nm. Then, experiments were divided into four sample groups: (a) CLMNPs-GFFG-FA, (b) CLMNPs-LGPV-FA, (c) CLMNPs-GFFG-FA-CPT, and (d) CLMNPs-LGPV-FA-CPT. In every group, eight aliquots of 100 μL of the diluted HRBC suspension were added in a series of centrifuge tubes containing (1) 1.9 mL of physiological saline solution as a negative control; (2) 1.9 mL of deionized water as a positive control; (3−8) 1.9 mL of samples at concentrations of 31.25, 62.5, 125, 250, 500, and 1000 μg/mL. After shaking gently and placing at a standstill for 3 h at room temperature, the eight aliquots of the mixtures in every group were centrifuged at 1500 rpm for 10 min, and the absorbance of the supernatants was recorded from 500 to 650 nm. The hemolysis percentages of the samples were calculated according to the equation below:

(Sample O.D.541−Negative control O.D.541) /(Positive control O.D.541− Negative control O.D.541) × 100%

Coagulation Assay in Vitro. Seven aliquots of 100 μL of fresh human plasma were added in centrifuge tubes containing 900 μL of physiological saline solution as a control, and a series of 900 μL of samples (CLMNPs-GFFG-FA, CLMNPs-LGPVFA, CLMNPs-GFFG-FA-CPT, CLMNPs-LGPV-FA-CPT copolymers) at concentrations of 31.25, 62.5, 125, 250, 500, and 1000 μg/mL. After shaking gently and incubating at 37 °C for 5 min, the mixtures were centrifuged at 2500 rpm for 10 min, and 700 μL of the supernatant was extracted by suction for analysis of prothrombin time (PT) and activated-partial-thromboplastin time (APTT) on a fully automatic blood-coagulation analyzer (ACL TOP 9000, USA) by using the SynthAsil kit (Instrumentation Laboratory Company, Orangeburg, USA). In Vitro Cytotoxicity Study. The cytotoxicity of the blank and the CPT-loaded CLMNPs-GFFG-FA or CLMNPs-LGPVFA copolymers was assessed by using the MTT assay. L02 cells and A549 cells (1 × 104 cells/well) were grown at 37 °C and under 5% CO2 atmosphere in folate-free RPMI-1640 medium in a 96-well plate, supplemented with calf serum (10%) and 1% penicillin−streptomycin in a fully humidified incubator. Then, the blank and CPT-loaded CLNMPs-GFFG-FA or CLNMPsLGPV-FA copolymers with a concentration of 25, 50, 100, 200, 300, 400, and 500 μg/mL, and the free CPT with a concentration of 1.23, 3.27, 6.53, 13.06, 19.59, 26.12, and 32.65 μg/mL were added to cell dishes, respectively, and then these cell dishes were put into incubators at 37 °C for 12 h. In order to demonstrate folate targeting, the cytotoxicity of the blank and CPT-loaded CLNMPs-GFFG or CLNMPs-LGPV copolymers with a concentration of 200 μg/mL against A549 cells was also measured. After incubation for a defined time, the culture medium was removed and 20 μL of MTT reagent (diluted in culture medium, 0.5 mg/mL) was added, followed by incubating for another 2 h. The MTT/medium was removed carefully and DMSO (150 μL) was added to each well to dissolve the formazan crystals. Absorbance of the colored solution was measured at 570 nm using a microplate reader (Bio-Rad, iMark). All experiments were performed in triplicate. Cell Imaging. After confirming the powerful fluorescence from two copolymers and no distinct autofluorescence from the cell itself under similar conditions, the cellular images were obtained with a laser scanning confocal microscope (LSCM, ZEISS, LSM 510 Meta, Germany). L02 cells and A549 cells (6 × 104 cells/well) were seeded on a 6-well plate at 37 °C for 24 h. After that, the blank and CPT-loaded CLNMPs-GFFG-FA or CLNMPs-LGPV-FA copolymers with a concentration of 200 μg/mL were added to the cell dishes, respectively. After a further 2 h incubation, these copolymer-loaded cells were washed with PBS three times to remove the free copolymers attached on the outer surface of cell membrane. Cell targeting was detected on LSCM for luminescence imaging under excitation wavelength of 488 nm. Cell imaging of three control experiments was also performed: L02 cells and A549 cells incubated with free CPT, and A549 cells incubated with CPTloaded CLMNPs-GFFG copolymer. All experiments were performed in triplicate. 1013

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RESULTS AND DISCUSSION Synthesis and Characterization of CLMNPs-Tetrapeptide-FA Copolymers. Synthesis of CLMNPs-tetrapeptide-FA copolymers consisted of the following three steps. In the first step, the CLMNPs were prepared for cellular imaging and magnetic targeting. First, the MNPs were treated with 5% aqueous ammonia to obtain MNPs with sufficient hydroxyl groups on the surface, which can facilitate coating MNPs with chitosan. Then, QDs photoactivated and MNPs were mixed together using the chitosan as a gelation agent according to ratios of 1/32/12 (chitosan/QD/MNP, wt/wt/wt) under sonication mode. Figure 2 show TEM images of Fe3O4 and

according to TEM. The QDs and the MNPs were randomly distributed all over the hybrid nanogel particle. The EDX analysis (Figure 3A) indicated that the elemental compositions of the CLMNPs were primarily Fe, O, Cd, Te, and N, which further confirmed the structure ingredients of the CLMNPs. On the basis of XRD pattern (Figure 3B(b)), the intensities of (311) Fe3O4 diffraction peaks from CLMNPs were weaker than those of pure Fe3O4 (Figure 3B(a), which manifested the presence of amorphous materials (chitosan) on the surface of Fe3O4. Furthermore, the FTIR spectra of the CLMNPs (Figure 3C(b)) showed that the characteristic peak at 1650 cm−1 corresponding to an amino group, two weak peaks at 2925 cm−1 and 1380 cm−1 corresponding to the stretching and bending vibration of methylene groups, and the broad peak situated at 1060−1030 cm−1 related to C−O stretching resulting from chitosan were observed, while these peaks were negligible in pure Fe3O4 MNPs (Figure 3C(a)). All these results verified that the chitosan-based luminescent/magnetic nanoparticles have been synthesized. In the second step, the end carboxyl groups of two kinds of tetrapeptide linkers (HOOC-GFFG-Fmoc and HOOC-LGPVBoc) were conjugated with the amino groups in chitosan molecules exposed to the CLMNPs surface by amido bond under activation of NHS and EDAC. After the intermediate products with blocking groups were subsequently processed by deprotection procedures, the CLMNPs-GFFG-NH2 and CLMNPs-LGPV-NH2 copolymers were obtained. On the b a s i s o f t h e F T I R s p e c t r a o f t w o c o p o ly m e r s

Figure 2. TEM images of (A) Fe3O4MNPs and (B) the as-prepared CLMNPs.

the as-prepared CLMNPs. It is clear that the CLMNPs tend to form a sphere in moderate sizes (about 140−160 nm)

Figure 3. (A) Representative EDX spectra of the CLMNPs-tetrapeptide-FA copolymer. (B) XRD patterns of the products prepared step by step. (C) FTIR spectra of (a) Fe3O4, (b) CLMNPs, (c) CLMNPs-GFFG, (d) CLMNPs-GFFG-FA, (e) CLMNPs-LGPV, and (f) CLMNPs-LGPV-FA. (D) The size distribution of the blank CLMNPs-tetrapeptide-FA measured by DLS. 1014

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Figure 4. (A) PL spectra of the as-prepared CLMNPs-GFFG-FA (a) and CLMNPs-LGPV-FA (b); the inset in right top shows the fluorescence microscope image of the CLMNPs-GFFG-FA copolymer (10×, excitation wavelength = 488 nm). (B) VSM magnetization curves of Fe3O4MNPs, CLMNPs, CLMNPs-GFFG-FA, and CLMNPs-LGPV-FA in applied magnetic field at 305 K. The inset in left top shows photograph taken under a 365 nm UV lamp: (a) CLMNPs, (b) CLMNPs-GFFG-FA, (c) Fe3O4MNPs, (d) TGA-capped CdTe QDs in deionized water. (b) and (c) are attracted by a permanent magnet. (C) Male S-180 sarcoma-bearing Kunming mouse being guided with a magnet after the CLMNPs-GFFG-FA copolymer was injected into the bloodstream by tail vein. (D,E) Representative fluorescence microscope images of the tumor slices indued without (D, control experiment) and with (E) magnet (10×, excitation laser wavelength = 488 nm). (F) Average value of PL intensity per microscopic field on the slice of control and magnet guidance.

than those of CLMNPs-tetrapeptide, confirming successful conjugation of folate to CLMNPs-tetrapeptide copolymers. Properties of CLMNPs-GFFG-FA/CLMNPs-LGPV-FA Copolymers. The PL spectra of CLMNPs-GFFG-FA and CLMNPs-LGPV-FA copolymer aqueous solutions at the same concentration (1 mg/mL) was shown in Figure 4A(a,b), respectively. The fluorescent CLMNPs-GFFG-FA and CLMNPs-LGPV-FA copolymers showed emission peaks at 565 and 572 nm when excited at 450 nm. According to the representative fluorescence microscopy image of the CLMNPsGFFG-FA (inset in Figure 4A), it is clear that highly fluorescent copolymers with orange−yellow emissions was prepared successfully. As shown in the VSM magnetization curves (Figure 4B), with step-by-step fabrication, the saturation magnetization declined accordingly due to the thickening of the shells. At the same time, two kinds of copolymers showed a similar saturation magnetization, which displayed negligible variation in magnetic properties although different precursors and operating steps were used in the course of synthesis. The saturation magnetization of two as-prepared copolymers (approximately 36 emu/g) accounted for about 45.6% of that of the MNPs (79 emu/g), reflecting an adequate magnetic responsiveness in the applications for magnetic targeting. According to the photographs of MNPs, CLMNPs, CLMNPs-GFFG-FA, and CdTe QDs in deionized water at 365 nm (inset in Figure 4B), the color conversion from bright yellow (pure CdTe QDs) to orange−yellow (CLMNPs-GFFGFA) showed the realization of effective reaction; at the same time, the achievement of magnetic copolymers with orange− yellow color fluorescence also verified success of conjugation. All the above results suggest that the end products, CLMNPsGFFG-FA and CLMNPs-LGPV-FA cpoplymers, integrated magnetic and fluorescent properties.

(Figure 3C(c,e)), a new characteristic peak appeared at 1660 cm−1, which indicated that the new bond of CONH2 groups linking tetrapeptides to the CLMNPs has emerged. Further, two absorption peaks at 1600 cm −1 and 1500 cm −1 corresponding to the stretching vibration of the aromatic ring skeleton derived from phenylalanine appeared in the CLMNPsGFFG-NH2 copolymer, while a distinct peak at 2950 cm−1 related to the stretching vibration of methyl groups derived from leucine and valine was present in CLMNPs-LGPV-NH2 copolymers. All the above results suggest that the two CLMNPs-tetrapeptide-NH2 copolymers have been successfully prepared. In the third step, to obtain CLMNPs-GFFG-FA and CLMNPs-LGPV-FA copolymers, the end amino groups on CLMNPs-GFFG-NH2 and CLMNPs-LGPV-NH2 were coupled with the γ-carboxyl group of folate by the amide bond. The coupling reaction was achieved in anhydrous DMF at room temperature in the presence of NHS and EDAC as the activating agent for the carboxylic group. The mean diameter of the CLMNPs-tetrapeptide-FA copolymer was determined to be about 150−190 nm by DLS (Figure 3D). Compared with the average diameters of CLMNPs based on TEM (Figure 2B), those of the CLMNPs-tetrapeptide-FA copolymers only increased by about 10−20 nm, reflecting that the agglomeration of copolymers is very little. Furthermore, the intensities of Fe3O4 diffraction peaks of the end products were weaker than those of the previous products based on XRD pattern (Figure 3B(b-f)), which indicated the presence of more amorphous materials (tetrapeptide and FA) on the surface of the copolymers. On the basis of FTIR spectra of the CLMNPsGFFG-FA and CLMNPs-LGPV-FA copolymers (Figure 3C(d,f)), two absorption peaks of benzene ring derived from folate at 1600 cm−1 and 1500 cm−1 were also stronger 1015

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Magnetic Targeting in Vivo. To estimate tumor target capability, we have also conducted in vivo copolymer accumulation experiments under magnetic attraction using male S-180 sarcoma-bearing Kunming mice (Figure 4C). As displayed in Figure 4D,E, the fluorescence imaging on tumor tissue slices indicates the presence of accumulated copolymers (dark orange fluorescence). The more intense fluorescence of magnetic copolymer on the magnet-field-exposed tumor slice (Figure 4E) is apparent as compared to the control slice (without magnet-field-exposed tumor) (Figure 4D). On the basis of the fluorescence distribution, it can be judged that some copolymers have even been transported into tumor cells by endocytosis. By further measurement for the fluorescence intensity on tumor tissue slices, the average PL intensity of the magnetic copolymers trapped in the tumor tissue per microscopic field was quite significant (Figure 4F), i.e., approximately 50 times greater with magnetic induction as compared to that for the control group sample. These results indicate that the magnetic copolymers were trapped successfully in the tumor tessue, even tumor cells under external magnetic field guidance. Drug Loading and Release. Under present experimental condition, the loading efficiency of CPT was 8.6 wt % for CLMNPs-GFFG-FA copolymer, namely, the actual loading amount of CPT on this copolymer reached approximately 70.2 mg/g. Moreover, CLMNPs-FA and CLMNPs-VPGL-FA were used to replace CLMNPs-GFFG-FA for further confirmation of the CPT-loaded mechanism. The results showed that the loading efficiency of CPT on CLMNPs-VPGL-FA copolymer was only 1.1 wt %, while there was nothing on CLMNPs-FA. Clearly, the binding site of CPT is right on the peptide linker, and the CPT loading efficiency has a significantly positive correlation with the hydrophobic degree of the peptide. In fact, CPT is conjugated to CLMNPs-GFFG-FA by means of both hydrophobic force and aromatic-ring stack force between CPT and phenylalanine in GFFG tetrapeptide. In the case of CLMNPs-FA, that is not appropriate for the CPT loading due to the hydrophilic chitosan surface of the CLMNPs. With respect to CLMNPs-LGPV-FA, although there was a length of LGPV tetrapeptide as linker on the chitosan shell, the CPT loading on CLMNPs-LGPV-FA only relied on the hydrophobic stacking force, and the binding force was relatively vulnerable. In addition, the traditional CPT-loading approach was to connect CPT to one end of the peptides through covalent bond.28,31 The present way was to combine CPT with side chain groups of the peptides, which prevented CPT from hydrolysis and toxicity diffusion to some extent by means of peptide pockets. This is a new concept having both drug loading and drug protecting. Figure 5 reveals the release behaviors of CPT-loaded CLMNPs-GFFG-FA and CLMNPs-LGPV-FA in a simulated normal body fluid (50 mM PBS, pH 7.4) and an acidic environment (50 mM PBS, pH 5.3) under dialysis condition at 37 °C. As shown in Figure 5, first, the CPT releasing results clearly demonstrated an initial burst release of CPT. For example, about 15% and 25% of CPT from CLMNPs-GFFGFA-CPT and CLMNPs-LGPV-FA-CPT copolymers can be released at the initial 1 h, respectively, while approximately ∼55% (pH 5.3) or ∼46% (pH 7.4) of CPT from the former and ∼69% (pH 5.3) or ∼57% (pH 7.4) of CPT from the latter can be released within 28 h. The initial burst release of the CPT may be related with the loading mode of CPT. Although the binding maintained by hydrophobic force may be unstable,

Figure 5. Release profiles of CPT from CPT-loaded CLMNPs-GFFGFA and CLMNPs-LGPV-FA copolymers in PBS solutions at pH 5.3 and pH 7.4 at 37 °C, respectively.

CPT was not readily removed by motionless washing. In fact, there were no differences in the release profiles after washing three times or more, indicating that the noncomplexed CPT molecules were nearly completely washed off before the releasing experiments. Therefore, we think that the initial burst release of the CPT should be triggered by shaking in a water bath. Second, the release efficiencies from two copolymers were higher at pH 5.3 than at pH 7.4. A possible reason may be that the quinoline ring (B ring) in the CPT molecule (insert in Figure 5) is extremely likely to generate positively charged salt under acidic condition (pH 5.3), which results in not only a decrease of binding force between CPT and peptide, but also mutual repulsion between CPT and the cation chitosan shell on CLMNPs. The another possible reason is the solubility of chitosan. At pH 5.3, chitosan polymer is probably soluble to water,17 so the water content of the shell would be high, and accordingly the repulsion would be more powerful due to hydrophobic effects of CPT; while at pH 7.4, chitosan polymer is insoluble, so the water content of the shell would be low, and accordingly the repulsion would be weaker. This is exactly what we expect, i.e., CPT could mainly be distributed around tumor tissues (acidic microenvironment) rather than located in a normal section. Finally, in comparison with the release capacity of CPT-loaded CLMNPs-LGPV-FA copolymer, that of the CPT-loaded CLMNPs-GFFG-FA copolymer is distinctly lower under similar pH condition, which may be attributed to higher binding force between CPT and the latter. Even so, the absolute release quantity of CPT from CPT-loaded CLMNPs-GFFG-FA was still significantly higher than from CPT-loaded CLMNPs-LGPV-FA, because the former possessed more CPT-loaded quantity. Blood Compatibility Assay. When materials are administrated by vein injection, excellent blood compatibility is a prerequisite to their applications in vivo, such as low hemolysis and coagulation effects.32 The investigation showed that no significant hemolysis effects were found for blank and CPTloaded CLMNPs-GFFG-FA/CLMNPs-LGPV-FA copolymers within the experimental concentration range (Figure 6A,B). The hemolysis percentages of both blank and CPT-loaded copolymers increased only slightly with an increased copolymer concentration in the range 31.25−1000 μg/mL. Even at a high concentration of 1000 μg/mL, as low as 1.66% and 1.75% hemolysis percentages were detected for CPT-loaded 1016

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Figure 6. Blood compatibility studies in vitro. (A) Representative absorption spectra in the supernatant of copolymer in PBS after incubation with HRBCs using water and PBS as the positive and negative controls. Inset on the right is a photograph of the hemolysis assay to detect the presence of hemoglobin in the supernatant of copolymer in PBS at the concentrations of 31.25, 62.5, 125, 250, 500, and 1000 μg/mL: (a) CLMNPs-GFFG-FA, (b) CLMNPs-LGPV-FA, (c) CLMNPs-GFFG-FA-CPT, (d) CLMNPs-LGPV-FA-CPT. (B) Hemolysis percentages of blank and CPT-loaded copolymers in PBS at above concentrations. (C,D) Coagulation properties of blank and CPT-loaded copolymers at above concentrations after incubation with fresh plasma for 5 min.

CLMNPs-GFFG-FA and CPT-loaded CLMNPs-LGPV-FA copolymers, respectively, and still fell within the negligible scope (