Study on Antheraea perny Silk Fibroin Nanoparticles Carried Insulin

Chinese oak tasar Antheraea pernyi silk fibroin nanoparticles are a promising biomaterial for drug delivery because of its good properties, such as biodegradability and biocompatibility. In recent decades, self-assembling nanoparticles derived from Antheraea perny silk fibroin have risen too much interest in application of drug delivery. In this paper, the Ca2+ in calcium gluconate induced self-assembling nanoparticles. It was fabricated by blending aqueous Antheraea perny silk fibroin solution, calcium ion and insulin in a proper mixing ratio. The structure of the silk nanoparticles carried insulin was characterized by X-ray diffraction, FTIR and DTA. The size and the morphology of silk nanoparticles were tested using scanning electron microscope and particulate size description analysers. The drug content, covering rate and in vitro cumulative release of silk nanoparticles were also determinate. The results demonstrated that the Ca2+ induced selfassembling nanoparticles hold the particle size of 200~500 nm. The phenomenon of the sustained release is comparatively obvious in the silk nanoparticles. To summarize, this research will open up a new experimental basis on the usage of macromolecular drug, insulin which can properly meet the satisfaction of the patients desired for sustained-release insulin.


Introduction
The gradual improvement of the living standard of human beings, the incidence of diabetes is increasing day by day. Although diabetes does not have the obvious symptoms of cardiovascular disease, the horror of AIDS and the intense rapid of cancer. Diabetes continues to erode human body. Diabetes acts an important role quietly among several major "killers" who endanger human life. And diabetes becomes the third largest diseases killer after cardiovascular diseases and cancer in threating to human health.
Silk fibroin has polypeptide chain segments, which presents random curl in dilute solution, α-helical structures in concentrated solution and water-insoluble β-hairpin formed while silkworms spin silk [1,2]. In the medical field, silk fibroin has good biocompatibility, biodegradability, and non-toxicity so that it can become a promising application of drug delivery medical materials.
Because of tryptophan, tyrosine and other amino acids, silk fibroin has a good antibacterial activity against mound, Staphylococcus aureus and Escherichia coli as well as the study on the skin sensitization in guinea pigs proves silk fibroin biocompatibility [3]. Historically, drug delivery systems are usually for oral or based injectable. However, medicines such as proteins or nucleic acids are considered as unsuitable for traditional treatment will face the dilemma. New technology is required to use in these new drugs to reduce side effects of these drugs, to optimize the efficacy of the drugs and enhance patient compliance. Recently the usage of nanotechnology has achieved the action to control release of new drugs. The nanotechnology can be applied in a wide range of small molecules, proteins or genetic drug delivery [4][5][6][7][8][9]. Drug release material needs to have excellent biocompatibility, biodegradability, low toxicity and appropriate mechanical properties. Chinese oak tasar Antheraea perny silk fibroin (ASF) is a natural polymer material, and having the ability to self-assemble, excellent mechanical properties, processing flexibility, good biodegradability and biocompatibility because of its unique structure and properties of silk fibroin [10].

Preparation of regenerated fibroin protein solution
At 98°C~100°C environment for 30 min, silk fiber was degummed by treating three times to extract sericin proteins and wax with an aqueous solution containing 5 g/L Na 2 CO 3 and 0.0625 g/L sodium dodecyl sulfate (SDS), bathing ratio 1:50, drying at 60°C. The degummed fiber was dissolved in a saturated solution of LiSCN at 50°C ± 2°C for 60 min, bathing ratio of 1:10. Then the solution was dialyzed in cellulose tube (14 KDa) against for distilled water for 4 d. The final concentration of aqueous solution was approximately 25 mg/mL then was placed in 4°C [11].

Morphology of drug-loaded ASF nanoparticles
The morphology of pure ASF nanoparticles and drug-loaded ASF nanoparticles was examined by via scanning electron microscopy (SEM, Hitachi S-4700, Japan) at an accelerating voltage of 15 kV. ASF nanoparticles were distributed in water by ultra-sonication, plated directly on a silicon plate and dried by vacuum later. The samples were coated gold sputter to prevent charging during SEM scanning. The size range and deviation of particles were examined by HPP 5001 laser diffraction scattering particle size analyzer (Malvern, UK).

Release of drugs from ASF nanoparticles
Ten milligrams of the drug-loaded ASF particles were subsequently re-dispersed in 10 mL Tril-HCl buffer in pH values of 5.2, 7.4 and 8.0 at 37°C to monitor the pH-dependent release. The suspensions were sampled at pre-determined time intervals of 0.25, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16 and 19 days. Next, the solutions were centrifuged at 12,000 rpm for 10 min to collect the solvent, and the nanoparticles were placed in fresh buffer solution. The equal free INS were dispersed in Tril-HCl buffer as the control. The percentage of cumulative release of insulin protein/FITC (%w/w) was measured using a FLS920 type steady-state fluorescence spectroscopy. The optical path length of the quartz cuvette is 1 cm. The experiment was repeated three times. The cumulative release rate was calculated using Equation (3).
amount of drug in the release medium Cumulative release rate (W/W%) 100% amount of drug loaded in the particles = × (3)

Structure of ASF nanoparticles
The pure ASF and drug-loaded ASF nanoparticles were frozen at -80°C and subsequently freeze-dried for X-ray diffraction (XRD) analysis. XRD analysis was conducted on an X 'PERT-PRO MPD Diffractometer (Panalytical Co., Holland) with a Cu-Kα radiation source. The scanning speed was 2°/min. The X-ray source was operated at a 30 kV voltage and 20 mA current. XRD patterns were recorded in the 2θ region from 5° to 45°.
The FTIR spectra were obtained using 5700 FTIR in the spectral region of 800 cm -1 to 1800 cm -1 . Respectively, freeze-dried ASF-INS nanoparticles (drug loading rate of 3.5%), ASF nanoparticles, a physical mixture of INS with ASF nanoparticles (3.5% weight of INS) and INS all mixed with KBr and then pressed into thin discs to be placed in the optical path directly to obtain infrared spectra of 800 cm -1 to 1800 cm -1 .

Thermal analysis
Studying crystal state and stability of the drug distribution in fibroin nanoparticles uses differential thermal analysis (Differential Thermal Analysis, DTA) with five milligrams of the

Results and Discussion
Embedding ratio and the drug content As shown in the Figure 1 the utilization ratio of ASF in ASF-INS nanoparticles changed linearly with the mass ratio of INS to ASF. When it is fixed the concentration of silk fibroin protein at 20 mg/ mL, it is seen that the utilization of ASF has a gradual downward trend from 98% to 88% with the increasing amount of INS. But the overall utilization of ASF remained at about 90% above. It proved ASF has been fully utilized and ASF will be an excellent material as a coating drug material.

Morphological analysis of ASF nanoparticles/ ASF-INS nanoparticles
The sizes and deviation range of ASF nanoparticles (Figure 4a)/ ASF-INS (Figure 4b) nanoparticles are measured by HPP 5001 laser diffraction scattering particle size analyzer (Malvern, UK).

Nano Research & Applications ISSN 2471-9838
As it can be seen in Figure 3, ASF nanoparticles and ASF-INS nanoparticles sizes range is between (200~400) nm. The average diameter of particles is about 300 nm with good dispensability.
The nanoparticles of ASF nanoparticles/ASF-INS nanoparticles were observed by S-4700 scanning electron microscope (SEM, Hitachi Company). Figure 4 is described that the nanoparticles are showing the size of uniform particle size in about 300 nm, regardless of ASF nanoparticles (Figures 5a and 5b) /ASF-INS nanoparticles (Figures 5c and 5d) which in line with the sizes measured by HPP 5001 laser diffraction scattering particle size analyzer. When the nanoparticles are enlarged to about sixty thousand times, the surface of the microspheres can be seen rough, uneven. This structure can increase the area of cell attachment, which would provide good conditions for the culture of cell in future.

Structure analysis of Nano drug-loaded particles
X-ray diffraction curve: X-ray diffraction curves were collected at different materials (Figure 6a). The 2θ mainly peaks at 16.7°, 20.20°, 24.90°and 30.9° nearby, corresponding to characteristic of the β-sheet. The mainly peaks represent α-helical crystalline structure close to 11.95°, 24.02° [13][14][15]. The main characteristics of silk fibroin solution conformation is α-helix and random coil structure [16,17]. And as we can be seen from Figure 6a, after fibroin microspheres formed by self-assembly, characteristic peaks attributed to α-helical structure disappeared, while it appeared more intense diffraction peak at 20.18°, moderateintensity diffraction peaks at 16.74° and 23.96° and a weak diffraction peak at 30.51°. It descripted that fibroin solution forms fibroin microspheres self-assembled, aggregation structure material changed to the β-sheet structure. Figure 6a (ASF-INS nanoparticles, 3.5%) was compared with Figure 6b (ASF nanoparticles freeze-dried samples) to find that they had the same peaks represented the same structure. The structure of the pure silk fibroin microspheres was consistent with of insulin, insulin tussah described self-assembled structure does not affect the nanoparticles. And diffraction peaks of b (ASF nanoparticles freeze-dried sample) and c (a physical mixture of INS with ASF nanoparticles (3.5% weight of INS) were compared with diffraction peaks of d (pure insulin) relatively large difference, indicating that pure insulin and insulin microspheres contained different structures, demonstrating good compatibility between insulin and fibroin.
Infrared spectroscopy: Since pure fibroin proteins exist mainly α-helix structure and random coil structure. The drawings ( Figure  6b) from a (ASF-INS nanoparticles, 3.5%), b (ASF nanoparticles freeze-dried samples) and c (a physical mixture of INS with ASF nanoparticles (3.5% weight of INS)) can be seen strong absorption peak at 1630 cm -1 and 1520 cm -1 , moderate absorption peaks at 1240 cm -1 and 964 cm -1 . Those peaks belong to β-sheet structure characteristic [18,19]. The aggregation structure of ASF by selfassemble into microspheres collapsed to the β-structural. This is consistent with the results of XRD. Figure 7 is DTG diagram derived from TG diagram. As it can be seen from the figure, with the increasing temperature, the quality of the materials decreased. The speed of reducing is rapid firstly and gentle then, between 250°C~400°C fastest. In Figure 7, d (pure insulin) obvious weight loss at 307.29°C, which is due to partial rupture between the molecule and a molecular chain of insulin breaking force. In Figure 7, a (ASF-INS nanoparticles, 3.5%) and b (ASF nanoparticles freeze-dried samples) apparent loss of focus at 350.60°C, which is the molecular chain of fibroin protein between local faulting and fracture caused by intermolecular forces [19,20] and the loss weight of c (a physical mixture of INS with ASF nanoparticles (3.5% weight of INS)) happened in the temperature between 307.29°C and 342.80°C, which shifts to lower significantly comparing to b (ASF nanoparticles freezedried samples). Explained that ASF-INS nanoparticles formed assembly having good stability and very compact structure is formed between insulin and silk fibroin.  release. At pH=8.0, the total release of the nanoparticles will be less than in a neutral environment up to 40%. Aim to solve the phenomenon, the nanoparticles were incubated in different pH environments, and the nanoparticles were removed after a period. ASF-INS nanoparticles dry samples were placed in different pH environments, and the results showed that their swells were very different (Figures 9a-9d). The size of ASF-INS nanoparticles ranges from 200 nm to 400 nm, and the average size is maintained at 300 nm with good dispersibility (Figure  9a). And they were placed in different environments having different swelling. The diameter of the nanoparticles was about 300 nm~600 nm and the average diameter was about 450 nm in pH=5.2 (Figure 9b). Insulin is a macromolecule protein drug, which has good biocompatibility with Antheraea perny silk fibroin and is not easy to be released from ASF nanospheres. Because the microspheres are very small under acidic conditions resulting in a slow release of INS from ASF-INS nanoparticles. The diameter of the nanoparticles was about 300 nm~700 nm and the average diameter was about 550 nm in pH=7.4, which resulted in the rapid release of INS from ASF-INS nanoparticles. After 6 days of release, the release rate gradually increased from the near-straight line to became stable (Figure 9c). The size of the nanoparticles was about (300~600) nm at pH=8.0 for 1d, the strongest particle size was at (400~500) nm and the average diameter was maintained at 500 nm. So that the amount release of INS in pH=8.0 is between the two above.

Conclusion
The particle size of nanoparticles studied in this experiment is between (200~400) nm and the average diameter of particles is 300 nm having dispersion coefficient and good uniformity.
When it is M INS /M ASF =12/100, embedding ratio is up to 28% and the drug content is 3.5%, which reaches good optimization of the comprehensive cost-effective ratio. In different environments, the release has greater rate gap owing to the unequal swelling conditions of ASF-INS nanoparticles. Under neutral conditions, it has faster release rate to release more than 50% after six days. Whether it will have the same influence for the all macromolecules or small molecule drugs, it will go on the further study. After some experiments about characterizing and testing the stability, it shows that INS has a good combination with ASF in ASF-INS nanoparticles by self-assembly. And ASF will be a promise material as a drug carrier.