Letter www.acsami.org
Controlled Antibiotics Release System through Simple Blended Electrospun Fibers for Sustained Antibacterial Effects Zixin Zhang,†,‡ Jianxiong Tang,§ Heran Wang,∥ Qinghua Xia,†,‡ Shanshan Xu,*,† and Charles C Han*,† †
State Key Laboratory of Polymer Physics and Chemistry, Joint Laboratory of Polymer Science and Materials, Beijing National Laboratory for Molecular Sciences, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, P. R. China ‡ University of Chinese Academy of Sciences, Beijing 100049, P. R. China § Huadong Hospital affiliated to Fudan University, Shanghai 200040, P. R. China ∥ Torch High Technology Industry Development Center, Ministry of Science and Technology, Beijing 100045, P. R. China S Supporting Information *
ABSTRACT: Implantation of sustained antibacterial system after abdominal surgery could effectively prevent complicated intra-abdominal infection. In this study, a simple blended electrospun membrane made of poly(D,L-lactic-coglycolide) (PLGA)/poly(dioxanone) (PDO)/Ciprofloxacin hydrochloride (CiH) could easily result in approximately linear drug release profile and sustained antibacterial activity against both Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli). The addition of PDO changed the stack structure of PLGA, which in turn influenced the fiber swelling and created drug diffusion channels. It could be a good candidate for reducing postoperative infection or be associated with other implant to resist biofilm formation.
KEYWORDS: electrospinning, bicomponent, control release, intra-abdominal infections, antibacterial activity
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layer as the diffusion obstacle,16 (iii) adding nanoparticles as adsorbent of drug molecules,23,24 (iv) coaxial electrospinning with shell as obstacle,15 (v) selecting model drug with different hydrophilicity,22 and (vi) modifying the interaction between polymer and drug by covalent bonds.25 However, these methods are either ineffective or with uncertain cytotoxicity. In this study, we intend to develop a sustained antibiotic release system utilizing U.S. Food and Drug Administration (FDA) approved polymers and broad-spectrum antibiotics to face safer degradation process and polymicrobial environment. Poly(D,L-lactic-co-glycolide) (PLGA) with controllable degradation rate and longtime clinical usage made itself an ideal drug carrier. Our previous study10 indicated that the addition of hydrophilic polymer could only induce more severe burst rather than sustained release. However, the release of antibiotics from monocomponent electrospun membrane still showed a little initial burst and unsatisfied release duration. We think that the molecular structure and swelling property of second component polymer may play a more important role in adjusting drug release profile. Therefore, three kinds of polymers, poly(ethylene glycol)-b-poly(D,L-lactic-co-glycolide) (PELA), polyglycolide (PGA) and poly(dioxanone) (PDO), which possess different molecular structure, hydrophilicity, and
ntra-abdominal surgery, such as abdominal wall hernias, gastrointestinal fistulas, and gastric perforation, may cause adhesion1,2 and infection3−6 in the postoperative surgical site, such as secondary acute peritonitis that are associated with substantial morbidity and mortality.3,5,7 A secondary operation is required if the infection is severe,8,9 and patients may die from toxic shock if the infection is not treated in time. To avoid the postoperative infection, aseptic operation is maintained and a heavy dosage of antibiotics is always applied by injection or oral in clinic. However, the antibiotics distributed to the rest of the body may cause severe side effects.4,10 Thus, sustained release of antibiotics for an adequate duration to the potential focal area is the key to prevent complicated intra-abdominal postoperative infection. Electrospinning create membranes with similar fibrous structure as the excellular matrix (ECM), which gives it excellent biocompatibility and ability as drug delivery system. And drug-loaded electrospun membranes could be used alone or be associated with other implant to prevent infections.11 Therefore, extensive investigations on electrospun membrane as desired delivery system have been carried out through combination of single-jet, multijets,12 coaxial jets,13−15 and multilayer10,16 processes. However, a few challenges remain unresolved, such as burst release,14,17 improper release duration,18,19 and uncompleted release.20,21 To research and solve these problems, a few methods were utilized, including (i) adding hydrophilic polymers to modify the hydrophilicity of the matrix,10,22 (ii) electrospinning multilayer film with the outer © XXXX American Chemical Society
Received: October 15, 2015 Accepted: November 24, 2015
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DOI: 10.1021/acsami.5b09820 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
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ACS Applied Materials & Interfaces
Figure 1. (A) Chemical structure of drugs and polymers. (B) Cross-section SEM micrographs (a−g) of membranes. The insets are LSCM and surface SEM micrographs.
monocomponent electrospun membranes were shown in Figure 2A to identify the influence of drug release from
degradation rate, were added as a second component, respectively. One of the three fluoroquinolone antibiotics, Mo (Moxifloxacin), MoH (Moxifloxacin hydrochloride), and CiH (Ciprofloxacin hydrochloride),18 which have a broad spectrum of antibacterial activity against both Gram-positive and Gramnegative pathogens, was selected as model drug. We expect that simple blend of proper polymers with antibiotics via electrospinning could achieve desirable release behavior and proper release duration. For example, electrospinning a drug carrying membrane which has a linear drug releasing profile in 7−14 days would exactly match the intra-abdominal infection treatment but require much less antibiotic usage. Besides, a combined medical device composed of FDA approved polymers and regular antibiotics could extremely decrease the registration time by FDA and avoid the safety risk in clinic use. To prepare electrospinning membranes, we dissolved PLGA (Figure 1A) and a second component polymer (PELA, PGA, or PDO (Figure 1A)) with a ratio of 93/7 w/w in hexafluoroisopropanol (HFIP), then a model drug (Mo, MoH, or CiH (Figure 1A)) with a concentration of 10% w/w of total polymer content was dissolved in the polymer solution. Finally, the electrospinning process was carried out in a sterile environment at 20 kV. Single PLGA or PDO with different drugs were also prepared. Uniform and smooth fibrous mats were successfully obtained for all membranes. Scanning electron microscope (SEM) micrographs showed that no obvious solid aggregates were observed on electrospun fibers. In the confocal laser scanning microscope (CLSM) study, intense fluorescence could be observed along the length of fibers in all of the membranes with drugs. Both the SEM and CLSM morphologies indicated that drugs were uniformly dispersed in fibers (Figure 1B). As the treatment duration for postoperative infection is normally no more than 14 days, the experiment period of this study was set as 360 h. The in vitro drug release profiles of
Figure 2. (A) In vitro drug release profiles of monocomponent electrospinning membranes with different drugs. (B) In vitro drug release profiles of bicomponent electrospinning membranes with the same drug. The samples were incubated in PBS buffer (pH 7.4) at 37 °C.
different polymers. With the increase in drug hydrophilicity (Mo < MoH < CiH), the drug release rate of monocomponent PLGA membranes slightly increased. The drug release profiles from PELA were not shown as PELA could not be electrospun into fibers due to its low molecular weight, as well as that from PGA whose initial drug releasing profile was similar to PLGA. However, the drug release dosage presented critically severe B
DOI: 10.1021/acsami.5b09820 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
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ACS Applied Materials & Interfaces
Figure 3. (a−g) Cross-section SEM micrographs of membranes after 7 days incubation. The insets are the surface SEM micrographs.
Figure 4. (A) Bacteria (S. aureus and E. coli) inhibition zone of drug-loaded membranes on agar plates after incubating specified time. (B) The S. aureus inhibition diameter (B1) and efficacy (B2) of drug-loaded membranes. (C) The E. coli inhibition area (C1) and efficacy (C2) of drug-loaded membranes.
film after incubation, and only top and bottom sides of the film formed drug diffusion channels. On the basis of this assumption, addition of semicrystalline PDO, amphiphilic PELA or faster degraded PGA should all increase the drug diffusion channel through decreasing the stack density of PLGA fibers or increasing the loose entanglement inner fiber, enhancing polymer hydrophilicity or accelerating polymer degradation. Significant difference was observed from the drug release profiles of bicomponent membranes as expectation (Figure 2B). A delightful drug release profile was observed from PLGA/PDO/CiH membrane, which is approximate constant release rate and 13 days release duration. Fibrous structure still could be observed after incubation in PBS for 7days (Figure 3e) which was somewhere between pure PLGA and pure PDO fibers. Meanwhile, PLGA/ PELA/CiH membrane showed small initial release dosage and hardly subsequent release amount, which should be contributed by the amphiphilic property of the second component PELA. The drug release from PLGA/PGA/CiH membrane occurred until PGA began to degrade in 5 days incubation. Neither of them were found fibrous structure at the cross-section after incubation (Figure 3f, g) indicated that neither of PGA or PELA with a ratio of 7% could stop the swelling of PLGA fibers from fusing together. Moreover, the similar hydrophilicity of PDO and PGA indicated no identified relationship between drug release rate and the hydrophilicity of the second component polymer. However, through understanding this mechanism of drug diffusion channel, the drug release dosage
increase when polymer carrier was changed to PDO. It can be seen from the inset of Figure 2A that drugs were completely released in about 12 h with a huge initial burst release. To further understand the key factors influencing the drug release profile, we characterized the surface and cross-section morphologies of the fibrous membranes by SEM after incubation for 7 days as shown in Figure 3. In the investigation period of this study, the degradation of main component PLGA did not cause weight loss of the membranes. Therefore, the drug release should be mainly controlled by fiber swelling and drug diffusion. However, after incubation in PBS for 7 days, the surface and cross-section morphologies of PLGA fibers changed substantially and fibers began to fuse together, no matter which drug was loaded. The intense swollen of PLGA fibers did not result in fast drug release. Instead, PDO fibers whose drug release rate was the fastest remained almost the same fiber diameter as those before incubation. The molecular chain arrangement in PDO fibers should be in more perfect order because of its crystallinity, compared to the amorphous polymer PLGA. The small crystalline structure in this semicrystalline polymer made PDO fibers fluffier than PLGA (Figure 1Be), especially after incubation (Figure 3d). The obvious fibrous structure and loose molecular chain entanglement inner fibers resulted in extremely high free drug diffusion channel on every fibrous surface, which consequently ended up with a burst drug release. However, the swelling of PLGA fibers is so strong that made the fibrous membrane into a nanoporous C
DOI: 10.1021/acsami.5b09820 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
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and duration can be easily controlled by changing the content of second component. More PELA related to more burst release. More PGA related to more supplementary drug release after its degradation. More PDO related to faster release rate and shorter release duration. The antibacterial activity of membranes related to antibiotics releasing dosage was also performed. S. aureus and E. coli were tested as Gram-positive and Gram-negative model bacteria. The intraperitoneal fluid would be completely replaced every 24 h. To simulate the intra-abdominal environment and situation, we observed the antibacterial behavior for 24 h at particular point, such as third to fourth day, rather than a period of cumulative release time, such as 0 to 10th day. In the first 24 h of antibacterial test, all samples with drugs present big inhibition zone and high antibacterial efficacy (Figure 4). The largest inhibition zone of PLGA/PELA/CiH at this moment was attributed to the highest burst release. However, after 4 days of drug release, only the inhibition zone and antibacterial efficacy of PLGA/PDO/CiH film still kept at a high level. On the other hand, as the drug release rate began to increase after 10 days because of membrane degradation, a profound shift of the inhibition zone and antibacterial efficacy happened to PLGA/ CiH, PLGA/PGA/CiH, and PLGA/PELA/CiH membranes again. This indicated that the sustained drug release behavior ensured its sustained long-term antibacterial activity. The antibacterial efficacy of PLGA/PDO/CiH membrane during the whole period of 15 days remained above 90%. The drug dosage loaded in fibers (about 10 mg in 10 cm × 10 cm fibrous membrane) was less than one percent of the dosage that oral or injected antibiotics treatment (CiH is around 0.2 g/day with need). This targeted implantation of loading drugs could extremely decrease the possible side effects. In conclusion, sustained antibacterial activity in agreement with drug release behavior was obtained. With proper ratio, PLGA/PDO/CiH membrane with sustained drug release behavior and duration about 13 days was developed by electrospinning, which is perfect for preventing postoperative infection. The addition of a second component with different structure changed the stack structure of PLGA, and resulted in different amounts of drug diffusion channels. This approach of designing a bi/multicomponent electrospinning membrane with the use of FDA approved polymers may help to develop functional drug release systems which can satisfy specific clinical requirements. Although PLGA/PELA/CiH and PLGA/ PGA/CiH membranes did not present sustained drug release with proper drug release rate, both of which have special characteristics in drug release profiles. The delayed drug release from PLGA/PGA/CiH membrane could be used for treatment of anaerobic bacteria, which usually occurred a few days after operation. The combination of PLGA/PDO/CiH, PLGA/ PELA/CiH and PLGA/PGA/CiH fibers by multijet or multilayer electrospinning may produce more possibilities for functional drug release systems to satisfy specific clinical requirements. These will be studied and reported in our future article.
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Letter
AUTHOR INFORMATION
Corresponding Authors
*E-mail:
[email protected]. Telephone: +86 82618089. Fax: +86 62521519. *E-mail:
[email protected] Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was financially supported by the National Nature Science Foundation of China (51003110) and the Knowledge Innovation Program of the Chinese Academy of Sciences (Grant KJCX2-YM-H19).
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S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.5b09820. Experimental details (PDF) D
DOI: 10.1021/acsami.5b09820 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
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