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Direct Formation of Silk Nanoparticles for Drug Delivery Liying Xiao, Guozhong Lu, Qiang Lu, and David L Kaplan ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.6b00457 • Publication Date (Web): 12 Sep 2016 Downloaded from http://pubs.acs.org on September 13, 2016

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ACS Biomaterials Science & Engineering

Direct Formation of Silk Nanoparticles for Drug Delivery Liying Xiaoa, b, Guozhong Luc, Qiang Lua, b,*, David L. Kapland

a

Collaborative Innovation Center of Suzhou Nano Science and Technology, Soochow

University, Suzhou 215123, People’s Republic of China b

National Engineering Laboratory for Modern Silk, Soochow University, Suzhou

215123, People’s Republic of China c

Department of Burns and Plastic Surgery, The Third Affiliated Hospital of Nantong

University, Wuxi 214041, People's Republic of China d

Department of Biomedical Engineering, Tufts University, Medford, MA 02155,

USA

Corresponding author: *

Qiang Lu, Tel: (+86)-512-67061649; E-mail: [email protected]

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ACS Paragon Plus Environment


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ABSTRACT：：Silk is useful as a drug carrier due to its biocompatibility, tunable degradation, and capacity in maintaining the function of drugs. However, further refinements are still required for silk-based nanoparticles to optimize applications as anticancer drug delivery systems. Here, a novel strategy was developed to prepare silk nanoparticles with improved performance. Unlike previous preparation methods that first obtain silk solutions and then induce nanoparticle formation through different treatments, here silk nanoparticles were directly prepared after a modified dissolution process. The nanoparticles had amorphous structure and homogeneous morphology, as well as improved dispersion in water and PBS solutions and improved pH-dependent drug release behavior when compared with the traditionally prepared silk nanoparticles. These improvements resulted in better uptake of the nanoparticles into cancer cells and higher cytotoxicity against cancer cells. These properties, when combined with the simpler and milder preparation process, indicate potential utility for anticancer drug delivery.

KEYWORDS: Silk; Drug Delivery; Nanoparticle; Anticancer; pH-responsive

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1. INTRODUCTION Silk is a versatile material system for sustained drug delivery because of its biocompatibility,

robust

mechanical

properties,

tunable

biodegradation

for

controllable drug release, utility in drug stabilization and aqueous-based fabrication and processing options for drug loading.1-4 Based on the requirements for different drug delivery applications, silk can be regenerated into various formats from injectable nanoparticles,5-10 microspheres,11-15 hydrogels,16-18 and adhesives, to films19-24 and scaffold25-29 implants. Further the structural hierarchy,8, 30 composition31, 32

and crystallinity,22, 24 of silk can be modified to match with the target drug to

achieve designed release behavior without the compromise of the drug activity. Different kinds of drugs including genes,14 biological drugs33-35 and small molecules36-38 exhibit controlled release from silk carriers since silk stabilizes various drugs as a function of entrapment, adsorption and encapsulation. The work to date suggests a promising future for silk-based drug delivery vehicles for clinical applications.37-41 Silk nanoparticles have attracted significant attention for anticancer drug delivery since different anticancer drugs can be loaded effectively and show higher release rate at lower pH conditions. Several strategies including phase separation,41 salting out,8, 32 emulsification,34 capillary microdot printing,41,

42

organic solvent precipitation,6,43

supercritical CO244 and freeze-induced self assembly

45-47

have been utilized to form

silk nanoparticles with different sizes. The anticancer properties of drug-loaded silk nanoparticles were confirmed with in vitro cell studies.5-9 The colloidal stability of

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silk nanoparticles was improved through coating with cationic polymers or crosslinking with polyethylene, which facilitated in vivo applications of the drug-loaded silk nanoparticles.5, 6 Though several critical improvements have been achieved, further modifications remain necessary since the present preparation processes are relative complex and use organic solvents.5, 6 Increasing pH-dependent release behavior for silk nanoparticles would also foster improved targeted therapy. Recently, novel solvent systems with weaker hydrogen-bond-destroying capacity were developed to maintain partial native structures of silk fibers in solution.48-50 Turbid gels quickly formed during dialysis in water or the removal of salts, suggesting the necessary of modifying the solvent systems to achieve silk materials with specific microstructures via aqueous processing. Here, the goal was to develop a new strategy to form silk nanoparticles directly by tuning solvent system based on the above approaches. Slightly higher LiBr concentrations were used to maintain some native nanostructures of silk fibers and restrain gel formation. After the usual dialysis and centrifugation processes that are used to prepare silk solutions, silk nanoparticles were formed directly. This approach led to better uniformity and metastable intermediate conformations of the nanoparticles. 2. MATERIALS AND METHODS 2.1. Preparation of the Silk Nanoparticles. Bombyx mori silk fibers were boiled in 0.02 M Na2CO3 for 20 min, and then rinsed thoroughly with ddH2O to extract the sericin proteins. Then a new combined solvent system composed of formic acid and lithium bromide was developed to control the

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degree of dissolution of the air dried fibers (Scheme 1). After serial experiments, degummed silk fibers were dissolved in an optimal combined solvent (weight ratio of silk, formic acid and lithium bromide (8 M): 1: 2.5: 33.3) at 60oC for 4 h, yielding a 2 w/v% solution. The solution was dialyzed against ddH2O using a dialysis tube (Pierce, molecular weight cut-off 3,500) for 3 days to remove the salt and acid. The resulting silk solution was centrifuged at 9,000 rpm for 20 min to achieve silk nanoparticle precipitates. After the supernatant was aspirated, the pellet was re-suspended in ddH2O to form milk-like silk nanoparticles (SNPs-F) solution. The concentration was then determined by weighing the solid state after drying.

Scheme1. The different models of silk nanoparticle formation processes: (A) New silk nanoparticle formation process where silk nanoparticles were directly prepared through dissolution without further steps. (B) Previous silk nanoparticle preparation processes which usually requires further treatments after solution formation. As a control, the silk nanoparticles were also prepared through organic solvent precipitation method43 and were termed as SNPs-A. Silk solution was first prepared 5



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according to previous procedures.28 The degummed silk fibers were dissolved in 9.3 M LiBr solution at 60oC for 4 h, dialyzed in water for 3 days, and finally centrifuged at 9,000rpm for 20 min to form aqueous silk solution. Then, 5 w/v% silk solution was added dropwise (150 µL/drop) to acetone, maintaining 85 v/v% acetone volume. Precipitated silk was then centrifuged at 100,000 × g for 30 min. After the supernatant was aspirated, the pellet was re-suspended in ddH2O, vortexed, and subsequently sonicated twice for 30 s at 20% amplitude with a SL-650D sonicator (Ultrasonic Cell Crusher, Shunliu, Nanjing, China). 2.2. The Size and Zeta-Potential Analysis of Silk Nanoparticles. Particle sizes and zeta-potentials of SNPs-F and SNPs-A were characterized by dynamic

light

scattering

(DLS,

Zetasizer

Nano-ZS,

Malvern

Instrument,

Worcestershire, UK) at 25oC. The concentration of samples for size and zeta-potential analysis was 250 µg/mL. Refractive indices of 1.60 for protein and 1.33 for ddH2O were taken for computation of particle size. Data were collected by measuring three independent sample batches. The stability of the particles in ddH2O was investigated after the particles were stored in ddH2O at 25oC for 0 and 28 days. Then the stability of SNPs-F and SNPs-A particles in culture media was assessed with DLS when the samples were dissolved in PBS (0.01 M pH 7.4) at 37oC for 0, 1 and 7 days without any preservatives. The morphology of samples was also confirmed with SEM (S-4800, Hitachi, Tokyo, Japan). 2.3. The Secondary Conformations of Silk Nanoparticles

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The secondary conformations were determined by circular dichroism (CD) spectra and fourier transform infrared spectroscopy (FTIR). CD analysis were performed with a spectrophotometer (Model 410, AVIV, Lakewood, USA) equipped with a Peltier temperature controller. Spectra were obtained from 250 to 190 nm at 25oC. The CD spectra represented the average of three measurements. FTIR spectra of freeze-dried silk nanoparticles were recorded on a NICOLET FTIR 5700 spectrometer (Thermo Scientific, FL, USA), equipped with a MIRacle ™ attenuated total reflection (Ge crystal). For each measurement, the wavenumber ranged from 400 to 4000 cm-1. 64 scans were co-added with a resolution of 4 cm-1. 2.4. Preparation of Doxorubicin (DOX)-Loaded Silk Nanoparticles. The as-prepared SNPs-F and SNPs-A solutions (500 µg/mL) were mixed with various concentrations of DOX (dissolved in water), followed by a 20 h adsorption period at room temperature under dynamic conditions. The DOX-loaded SNPs-F were collected by 20 min centrifugation at 9,000 rpm, while DOX-loaded SNPs-A were collected by centrifugation at 20,000 × g for 20 min. The amount of drug remaining in the solution was determined by measuring DOX-associated fluorescence (excitation 480 nm, emission 590 nm) using a Hitachi UV 2910 UV-vis spectrometer. The loading efficiency was calculated according to the following formula: Loading efficiency (%) =

W T − WF ×100% WT

where WT is the total weight of DOX in the reaction, WF is the total weight of free DOX remaining in the supernatant.

2.5. In Vitro Release of DOX-Loaded Silk Nanoparticles. 7



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The DOX-loaded nanoparticles (2.5 mg) were dispersed in 2.5 mL of PBS at pH 4.5, 6.0, and 7.4, respectively. Since it was hard to separate the released DOX drugs from that still loaded on the nanoparticles in the system, the dispersed samples were loaded into a Slide-A-Lyzer dialysis cassette (MWCO 3,500) and inserted into 5 mL of PBS with same pH value. The samples were incubated at 37°C in a shaker for 8 days to investigate drug release. At predetermined time points, the DOX in the medium solution outside the cassettes was monitored using fluorescence spectroscopy (excitation 480 nm, emission 590 nm).The PBS buffer outside the cassettes was replaced with the same volume (5 mL) of fresh PBS, maintaining sink conditions throughout the experiment. The release measurement was repeated triplicate for every sample. DOX-free silk nanoparticles were also investigated under the same conditions and failed to find silk outside the cassettes, confirming that DOX-loaded silk nanoparticles could be blocked by the cassettes.

2.6. Labeling Silk Nanoparticles with FITC 25 mg of FITC (fluorescein isothiocyanate) in 5 mL of DMSO was slowly added to 5 mL of 500 µg/mL silk nanoparticles suspension in borate buffer (50 mM, pH 8.5) and allowed to react for 2 h at room temperature in the dark while stirring. FITC labeled SNPs-F were then recovered by 20 min centrifugation at 9,000 rpm, while FITC labeled SNPs-A were collected by centrifugation at 20,000 × g for 20min. To remove the unconjugated FITC, the labeled nanoparticles were subjected to repeated cycles of washing with PBS (pH 7.4) and centrifugation until no fluorescence (excitation 490 nm; emission 520 nm) was detected in the supernatant. Finally, the

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FITC labeled silk nanoparticles were dialyzed against ddH2O for 3 days in the dark and processed for in vitro cell culture studies as detailed below.

2.7. Cell Culture. The human breast cancer cell line MCF-7 was obtained from the cell bank of Chinese Academy of Science (Shanghai, China). Bone marrow mesenchymal stem cells (BMSC) were derived from SD rats. The cells were cultured in DMEM, supplemented with 10% (v/v) FBS, 100 U/mL penicillin, and 100 µg/mL streptomycin at 37°C using a humidified 5% CO2 incubator.

2.8. In Vitro Trafficking of Silk Nanoparticles. MCF-7 cells were plated at a density of 2 × 104 cells/well in 24-well glass bottom plates and allowed to recover for 24 h. Next, the cultures were incubated for 2 h with 500µg/mL FITC-labeled silk nanoparticles. The media was removed and washed several times with PBS, followed by refilling the wells with 1 mL of fresh complete DMEM medium containing Lysotracker Red (Invitrogen, Grand Island, NY, USA). After 0.5 h incubation, the media were removed and washed several times with PBS, followed by refilling the wells with 1 mL of fresh medium. Finally, the cells were imaged using laser scanning confocal microscope (LSCM, Olympus Fluoview FV 1000, Japan).

2.9. In Vitro Cytotoxicity Study. The cytotoxicity of DOX-free SNPs-F and SNPs-A was assessed with MCF-7 and BMSC cells. The cells were seeded in 96-well plates (1.9 × 104 cells/cm2) with DMEM-FBS medium and incubated at 37°C with 5% CO2 for 24 h. After removing

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the medium, 200 µL of DMEM-FBS containing various concentrations of SNPs-F or SNPs-A was added, and incubated for 3d. The standard cell viability CCK-8 assay was then carried out.43 To study the cytotoxicity of DOX-loaded nanoparticles, MCF-7 cells (1.9 × 104 cells/cm2) were plated in 96-well plates with DMEM-FBS and recovered for 24 h. After removing the medium, 200 µL of DMEM-FBS containing various concentrations of free DOX, DOX-loaded SNPs-F and DOX-loaded SNPs-A was added, respectively, and incubated with the cells for different periods of time. The standard cell viability CCK-8 assay was then carried out.6

2.10. Statistics. Sample pairs were analyzed with the Student’s t-test. Multiple samples were evaluated by one-way ANOVA analysis followed by Bonferroni’s multiple comparison post hoc test. Measurements are presented as mean ± standard deviation, unless otherwise specified. Results were considered to be statistically significant at *P ≤ 0.05, **P ≤0.01.

3. RESULTS AND DISCUSSION 3.1. Silk Nanoparticle Generation and Characterization Except for the solvent system, the SNPs-F preparation processes were similar to the silk dissolution process described previously.48-50 A combined solvent system composed of formic acid and lithium bromide with specific concentration was carefully tuned to control the degree of the hydrogen bond destruction among silk fibroin molecules. After the removal of salt and acid through dialysis, the SNPs-F

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particles were collected directly through centrifugation (Scheme 1) and the particles easily dispersed in water to form a milk-like aqueous solution (Fig 1Aa, insert). As a control, silk nanoparticles were also prepared through organic solvent precipitation,43 and were termed as SNPs-A. For previous silk particle preparation processes, obtaining silk aqueous solution is a prerequisite and then various further treatments are required for nanoparticle formation. Here, a more controllable, milder and simper process was developed (Scheme 1). The characterization of SNPs-F particles was then investigated. The SNPs-F particles presented a spherical geometry without obvious aggregation (Fig 1). The size of the particles was 100-200 nm, slightly higher than that of SNPs-A. Significant bigger size of SNPs-A particles appeared in DLS curves, suggesting that the SNPs-A particles aggregated in aqueous solution. Compared with SNPs-A, the SNPs-F particles achieved improved uniform morphology and better dispersability, which was further confirmed by DLS. Since homogeneous particles usually exhibit more uniform release behaviors than polydisperse particles, the improved dispersion should improve reproducibility of release behavior for drug delivery.34 Different preparation processes also resulted in changes of surface charge and secondary conformation.10 Zeta potential measurements were used to study surface charge of the different silk nanoparticles. The SNPs-F particles had significantly lower charge density than the SNPs-A particles, suggesting reduced electrostatic repulsive forces between the SNPs-F particles (Figure 1Bb).This result is counter-intuitive since repulsive force is a critical factor of restraining particle aggregation. Therefore, the secondary structures

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were then clarified with CD (Figure S1) and FTIR spectra (Figure 1Bc). The secondary structure of the SNPs-A particles was dominated by β-sheets while the SNPs-F presented mainly amorphous features. Thus the SNPs-F particles with lower charge density likely showed lower aggregation due to their amorphous state, providing better dispersion in aqueous solutions when compared to the β-sheet-containing particles with hydrophobic features.11

Figure 1. The characterization of SNPs-F and SNPs-A particles: (A)The state and micromorphology of SNPs-F (a) and SNPs-A (b); (B) The size distribution (a), zeta potential (±SD; n ≥ 3) (b) and FTIR (c) of the different silk nanoparticles. The stability of the particles was further assessed after the particles were stored for up to 28 days in H2O at 25oC (Figure 2A). Although both the nanoparticles showed size increases based on DLS results (Figure 2Ab), the SNPs-F particles had better stability when compared to the SNPs-A particles. The SEM images (Figure 2Aa) showed that the SNPs-F particles maintained their spherical shapes without significant 12


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aggregation while the SNPs-A particles aggregated and lost their original morphology. The nanofibers with lengths of several hundred nanometers and diameters of about 20 nanometers appeared in the SNPs-F samples after the 28 day culture, resulting in the size changes in DLS results. Based on our previous study,51 the nanofibers should be released from the silk nanoparticles without impacting the dispersion of silk in water. The particles were then exposed to PBS to assess the stability and aggregation of SNPs-F and SNPs-A particles in culture media (Figure 2B). After 7 days, serious aggregation appeared for the SNPs-A, resulting in precipitation. Unlike the SNPs-A, the SNPs-F solution changed from milky to a transparent state, suggesting that silk materials with smaller sizes rather than aggregates formed.37 SEM images (Fig 2Bb) confirmed that silk nanofibers were released from the SNPs-F nanoparticles and maintained the nanofiber morphology without aggregation in the PBS solution, while the SNPs-A particles aggregated to form larger particles. Recently, the colloidal stability of silk nanoparticles was improved through coating with a cationic polymer.6 The coated silk nanoparticles maintained enough stability in biological media without sacrificing drug loading and delivery capacity.6 Compared to the coated silk nanoparticles, the SNPs-F nanoparticles directly prepared through dissolution process showed improved stability and smaller sizes in biological environments.

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Figure 2. The stability of the different silk nanoparticles: (A) The stability of the SNPs-F nanoparticles (A1) and SNPs-A nanoparticles (A2) in H2O at 25 oC: (a) SEM images of the particles after cultured in H2O for 28 days, (b) the size changes of the particles at 0 and 28 day; (B) The stability of the SNPs-F nanoparticles (B1) and SNPs-A nanoparticles (B2) in PBS solution at 37oC: (a) macrograph state of particles after cultured for 7 days, (b) SEM images of the particles after cultured for 7 days, (c) the size changes of the particles at 0, 1 and 7 day.

3.2. Drug Loading and Release from the Silk Nanoparticles The drug loading and encapsulation with these silk nanoparticles were examined. Since it is well known that silk facilitates the adsorption of weakly basic drugs,3, 10

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doxorubicin (DOX), an anticancer drug, was used to test the loading characteristics of the silk nanoparticles. The DOX-loaded silk nanoparticles remained the nanosphere morphology without significant size changes (Figure S2), confirming their stability in pure water. Using 500 µg of the silk nanoparticles, the loading characteristics of DOX was studied (Figure 3A). The percentage of bound drug gradually declined following an increase of DOX/silk ratios (w/w). Compared to that of SNPs-A, lower drug levels were absorbed on the SNPs-F particles at lower DOX/silk ratios, and then became similar when the ratio was in the range of 0.3-0.6. The loading efficiency was comparable or significantly higher than previous silk particle systems.43, 47 The total amount of loaded drug increased and remained constant at about 50 µg after above 200µg DOX was added in aqueous solutions containing 500µg silk nanoparticles, suggesting saturation. Several studies have confirmed that the DOX loading capacity of silk particles was mainly dependent on negative charge and hydrophobic properties.5, 31 Thus it is reasonable that the SNPs-F particles had a lower loading percentage at lower ratios. The potential loading capacity of the SNPs-F particles was developed following the increase of DOX amount in the system, finally achieving similar saturated loading of DOX.8 Other model materials including tetramethyl rhodamine conjugated bovine serum albumin, tetramethyl rhodamine conjugated dextran and rhodamine B were also loaded on the silk nanoparticles via similar loading process, suggesting promising applications in various drugs. Further development will be continued in our following studies. Next, the release behavior of DOX from the particles was examined at different pHs (Figure 3B). The DOX-loaded

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SNPs-F and SNPs-A particles prepared at the DOX/silk ratio of 0.5 were used as model due to their similar loading percentage of DOX. The PBS buffers with three different pH values (pH 7.4, 6.0 and 4.5) were used to mimic those of blood plasma, early endosomes and lysosomes, respectively.43 Both of the two particles showed similar release behavior at the different pH values. A burst release was observed in the first 18 h followed by sustained release for the next 8 days. A pH dependent release behavior was observed for both the particles, which showed fastest release rate at pH 4.5 and slowest at pH 7.4. An improved pH sensitive property was achieved for the DOX-loaded SNPs-F particles. In the neutral environment, the release rate of DOX from the SNPs-F particles was significantly slower than that from the SNPs-A particles, achieving 12 and 16% after 8 days for the two particles, respectively. However significantly faster release behavior was observed for the SNPs-F particles in acidic conditions (pH 6.0 and 4.5). About 27 and 44% of the loaded DOX was liberated after 8 days at pH 6.0 and 4.5 from the SNPs-A particles while the amounts increased to 31 and 46% for the SNPs-F particles.

Figure 3. DOX loading capacity on the silk nanoparticles and the release behaviors of the loaded DOX: (A) Loading efficiency at different DOX/silk ratios; (B) DOX

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release behavior from the SNPs-F and SNPs-A silk nanoparticles at pH 7.4, 6.0 and 4.5 (*P≤0.05, **P≤0.01, ±SD, n = 3).

The SNPs-F particles were cultured in the PBS solutions with various pH values to reveal the reason for the better pH sensitivity (Figure 4, S3). At pH 7.4, the conformational transitions of the SNPs-F particles were restrained while the nanoparticle structures of the SNPs-F were destroyed and transformed into nanofibers in PBS solution within 1 h (Figure 4Ac, f), which might result in the increase of surface area.52 Following the decrease of pH, the nanostructural transitions were restrained and the amorphous structures were maintained while more intermediate conformations formed, achieving the preservation of nanoparticle structure and amorphous state at pH 4.5. A similar phenomenon was found in our recent study and could be explained by the balance of the negative charges inside and outside silk aggregates.51 Metal ions usually act with functional groups containing negative charges outside the aggregates while hydrogen ions could neutralize the negative charges both outside and inside of the aggregates simultaneously due to their different capacity of entering into the aggregates. When only the negative charge outside silk aggregates was shielded, the repulsive force of the negative charge inside the aggregates could result in the destruction of the aggregates. Here, since the balance of the negative charge was destroyed at pH value of 7.4, the amorphous silk nanoparticles were destroyed to form small nanofibers. The surface area of silk could increase after the above transition, which resulted in stronger interaction with DOX.53 The loaded DOX on the SNPs-F particles released more slowly under neutral 17



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conditions. In the acidic PBS solutions, since the SNPs-F particles could maintain their original structures that showed weaker DOX-loading capacity than the SNPs-A particles, higher release rates resulted.

Figure 4. The morphology and secondary conformation of SNPs-F particles in PBS solutions with various pH values: (A) The morphology images of SNPs-F particles at pH 4.5 (a, d), pH 6.0 (b, e) and pH 7.4 (c, f), respectively; (B) The size and secondary structure changes of the SNPs-F particles: (a) DLS results and (b) FTIR spectra. Before the investigation, the samples were cultured in PBS solution with various pH values at 37 oC for 1 h.

3.3. Cellular Uptake of the Silk Nanoparticles The FITC-labeled silk nanoparticles were cultured with MCF-7 cells to study cellular uptake. After incubation with nanoparticles for 2 h, most of the SNPs-A

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particles separated from the cells while the SNPs-F particles had been internalized by the cells (Figure 5). This phenomenon indicated that the SNPs-F particles could be taken up more easily by the cancer cells. Several studies have confirmed that smaller size of the nanoparticles could facilitate cellular uptake.5, 31 Although further study was necessary to elucidate the differences in cellular uptake efficiency, the stability of the particles in aqueous environments could be a critical factor since the SNPs-A particles easily aggregated into larger particles while the SNPs-F particles maintained good colloidal stability and even formed smaller nanofibers under the same conditions.

Figure 5. LSCM images of the MCF-7 cells cultured with SNPs-F and SNPs-A nanoparticles. Green, FITC-labeled silk nanoparticles; Red, lysotracker-labeled lysosome. Scale bars, 20 µm.

3.4. In Vitro Cytotoxicity Studies The cytotoxicity of free silk nanoparticles was assessed using MCF-7 and BMSC cells (Figure 6A). Both the blank silk nanoparticles showed negligible cytotoxicity. 19



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Compared to the SNPs-A particles, the SNPs-F particles showed lower cytotoxicity. The MCF-7 cells demonstrated > 90% viability when the concentration of the SNPs-F particles was increased to 125 µg/mL, while showed only > 90% and 80% viability at 10 µg/mL and 125 µg/mL of the SNPs-A particles, respectively. The BMSC cells showed > 90% viability when cultured with the SNPs-F particles at concentration of 125 µg/mL, but the viability decreased to > 80% for the SNPs-A particles at same concentration. The possible reason was that the SNPs-F nanoparticles were prepared via a simpler and milder all-aqueous process, which avoided possible residual organic solvent that can occur for the SNPs-A particles. Next, the response of MCF-7 cells to the DOX-loaded nanoparticles and free drug using an equivalent DOX dose was investigated by the CCK-8 assay (Figure 6B). At the first 24 hours, compared to DOX-loaded SNPs-A and SNPs-F particles, the free DOX exhibited higher cytotoxicity to the cancer cells at the same dose of DOX, since more free DOX could be taken up quickly by passive diffusion.6,

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The free DOX still showed higher

cytotoxicity than the DOX-loaded SNPs-A particles in the following period, possibly since the aggregation of the particles restrained effective uptake by the cells.6 Interestingly, when the cells were exposed to the DOX-loaded SNPs-F particles for above 24 hours, cytotoxicity was significantly improved compared to the equivalent amount of free drug, suggesting suitable stability as well as improved pH sensitivity of the SNPs-F particles resulting in the higher cytotoxicity. These results indicated that the silk nanoparticles prepared via this simpler and milder process achieved improved performance for anticancer drug delivery in vitro.

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Figure 6. In vitro cytotoxicity studies of free and DOX-loaded silk nanoparticles: (A) The cytotoxicity of free SNPs-F and SNPs-A silk nanoparticles against MCF-7 (a) and BMSC cells (b) when cultured for 3 days; (B) The cytotoxicity of DOX-loaded SNPs-F and DOX-loaded SNPs-A against MCF-7 cells when cultured for 6, 12, 18, 24, 48 and 72 h. Cell viabilities of free DOX, DOX-loaded SNPs-F and DOX-loaded SNPs-A at different DOX concentrations (4 µg/mL; 1 µg/mL) were examined as a function of incubation time via CCK-8 assay. Significant differences were determined with Student’s t test, *P≤0.05, **P≤0.01, ±SD; n≥3.

4. CONCLUSIONS Silk nanoparticles were directly generated by tuning the dissolution solvent system. These silk nanoparticles had amorphous states and suitable stability in aqueous and PBS solutions, avoiding aggregation that can occur for silk particles reported previously. Higher cell uptake and improved pH sensitivity were also achieved with

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these particles, resulting in improved cytotoxicity for cancer cells in vitro when delivering DOX. These results establish a novel strategy to develop more advanced silk-based nanomedicines through easier and milder processes.

ASSOCIATED CONTENT

Supporting Information

CD spectra of SNPs-F and SNPs-A particles; SEM image of DOX-loaded SNPs-F particles and CD spectra of SNPs-F particles in pH 4.5, pH 6.0 and pH 7.4 PBS solutions.

ACKNOWLEDGEMENTS

The authors thank National Basic Research Program of China (973 Program, 2013CB934400), NSFC (21174097, 81272106) and the NIH (R01 DE017207, P41 EB002520). We also thank the Excellent Youth Foundation of Jiangsu Province (BK2012009) for support of this work.

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