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J. Biophotonics 4, No. 6, 403–414 (2011) / DOI 10.1002/jbio.201000121
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Dynamic study of PLGA/CS nanoparticles delivery containing drug model into phantom tissue using CO2 laser for clinical applications Mahboobeh Mahmoodi*; 1; 2 , Mohammad E. Khosroshahi*; 1 , and Fatemeh Atyabi 3 1 2 3
Amirkabir University of Technology, Faculty of Biomedical Eng., Biomaterial Group, Laser and Nanobiophotonics Lab., Tehran, Iran Islamic Azad university of Yazd, Faculty of Eng., Material Group, PO Box 89195/155, Yazd, Iran Novel Drug Delivery Systems Lab, Faculty of Pharmacy, Medical Sciences, University of Tehran, PO Box 14155-6451, Iran
Received 21 December 2010, revised 20 January 2011, accepted 22 January 2011 Published online 16 February 2011
Key words: superlong CO2 laser, chitosan, fast photography, photothermal deflection, laser drug delivery, nanoparticle
In this study, cationic nanoparticles (NPs) were prepared by coating chitosan (CS) on the surface of PLGA NPs. To our knowledge most of the work in the field of drug delivery systems using lasers has been performed using short pulses with micron and submicron durations. We carried out an experiment using superlong PLS-R (10 ms) and CW CO2 laser modes on simulated drugbiogelatin model where drug was encapsulated by PLGA/CS NPs. Maximum depth of drug containing cavitation was achieved faster at higher powers and shorter irradiation time in CWC mode. We believe that the main mechanism at work with superlong pulses is both photothermal due to vaporization and photomechanical due to photophoresis and cavitation collapse. In the case of CW, however, it is purely photothermal. Thus, drug molecules can be transported into tissue bulk by thermal
1. Introduction To date, most of the advanced nanoparticulate (NPs) drug carriers have been developed by utilizing either synthetic or natural polymers or by their combination. For example, PLGA (Poly lactide-co-glycolide acid) is extensively used in biomedical and pharmaceutical applications. PLGA is hydrolytically unstable and insoluble in water; it degrades by hydrolytic attack of ester bonds [1]. Among the various natural polymers available, CS is perhaps one of the
Time resolved laser induced cavitation at 3 W and 5 Hz. waves which can be described by the Fick’s law in 3-D model for a given cavity geometry and the mechanical waves, unlike only by pure photomechanical waves (i.e. photoacoustically) as with short pulses. Therefore, our studies could offer an alternative for currently existing method for drug delivery.
most widely used biopolymers for the preparation of NPs [2, 3]. CS is a weak base and is insoluble in water and organic solvents, however, it is soluble in dilute aqueous acidic solution with pH < 6.5. Particle size, density, viscosity, degree of deacetylation, and molecular weight are important characteristics of CS which influence the properties of pharmaceutical formulations based on CS. Drug delivery systems (DDS) are an area of study in which researchers from almost every scientific discipline can make a significant contribution.
* Corresponding authors: e-mail:
[email protected], Phone: +989121852480, Fax: +983517222043;
[email protected], Phone: +9821-64542398, Fax: +9821-66468186
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BIOPHOTONICS 404
M. Mahboobeh et al.: Synthesis and dynamic study of DR-PLGA/CS nanoparticle delivery using CO2 laser
Understanding the fate of drugs inside the human body is a high standard classical endeavor, where basic and mathematical analysis can be used to achieve an important practical end. No doubt the effectiveness of drug therapy is closely related to biophysics and physiology of drug movement through tissue. Therefore, DDS requires an understanding of the characteristics of the system, the molecular mechanisms of drug transport and elimination, particularly at the site of delivery. In chemical methods, cationic lipids, polymers and liposomes can be used as a drug carrier [4, 5] while physical methods such as high voltage electric pulse [6], CW ultrasound [7], extra corporeal shock wave [8, 9], laser induced shock wave [10, 11] have been used as a driving force for drug delivery. In transcutaneous laser injection, drug solution is applied onto skin surface topically and then it is irradiated by a laser pulse. Strong absorption of IR lasers (e.g. Er : YAG or CO2) irradiation by water leads to perforation of both drug solution and skin tissue [12, 13]. In our first report on laser induced cavitation and the role of photoacoustic effects [14] we showed that hot, high-pressure vapor cavity produced at 2.6–3 mm can lead to energy being transported well beyond the laser beam penetration depth as a direct result of the bubble expansion following the pulse and large amplitude acoustic waves associated with bubble formation and decay. Since then many efforts have been reported on the optical cavitation dynamics [15–20] and potential use of cavitation and photoacoustic [21] as a means of delivering drug into tissue [22–30] and generally on a solid boundary [31–33]. However, for more accurate and localized delivery, controlled drug release based on nanotechnology has been one of the recent developments in this field. To our knowledge no specific work was found in the literature regarding the dynamic studies of long chopped pulse (tp ms) and CW CO2 laser interaction with drug NPs-biogelatin model as a method of drug delivery. With this view we have used fast photography and photothermal deflection technique to gain an insight into the mechanism of mass transfer into the bulk medium. Also, in this study, Direct Red-encapsulated PLGA and PLGA/CS were fabricated and NPs have been characterized in terms of particle size and encapsulation efficiency and photomechanical drug delivery.
2. Materials and method 2.1 PLGA-encapsulated Direct Red NPs Nanoparticles were fabricated via the W/O/W double emulsion solvent evaporation surface coating method, as previously described [34]. Briefly, 3 ml of de-
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ionized aqueous 0.18% Direct Red 81 (drug model) (Sigma Alderich-USA) was poured into 15 ml of dichloromethane solution (DCM) (Merck, Germany) containing 300 mg of PLGA (50 : 50, Resomer RG 504 H, Mw 48000, Bohringer Ingelheim, Germany), and then emulsified using a sonicator (Tecna6-TecnoGA2-S.P.A.) to form a W/O emulsion. The W/O suspension was added to 30 ml of 1 wt% of polyvinyl alcohol (PVA; Mw 22000, Merck), and emulsified using the same sonicator to produce W/O/W emulsion. 300 ml of 0.5 wt% of PVA was added to the emulsion which was mechanically stirred. The suspension was evaporated for 18 h and stirred at 250 rpm to remove the solvent from the emulsified suspensions. The suspension that contained PLGA-encapsulated Direct Red NPs was centrifuged (Sigma, 3K30, RCF 25568, speed 16500 with rotor 12150 H, Germany) for 20 min to separate the NPs from the suspension. The NPs were rinsed with distilled water and then centrifuged several times to remove PVA and residual solvent. The NPs were further filtered through membrane filters to remove large sub-micron particles. Then, the NPS dried at a freeze dryer (Chaist, Alpha 1–2 LD plus, Germany) for storage.
2.2 CS-coated PLGA-encapsulated Direct Red NPs To prepare the CS solution (low molecular, 80–85% deacetylation, Merck) for this work, 300 mg CS was dissolved in 150 ml of 1% acetic acid solution and similarly PLGA NPs was followed, except that 150 ml of the 0.1 wt% CS solution and 150 ml of 0.5 wt% PVA solution were added, instead of 300 ml PVA 0.5% solution, to the aforementioned W/O/W emulsion with continuously stirring. The pH value of the emulsion was adjusted to 6–7 during solvent evaporation to enhance CS coating onto PLGA NPs to yield PLGA/CS NPs.
2.3 Preparation of biogelatin In this study, the tissue was modeled using commercial biogelatin (3.5% Gelatin-175 Bloom-Sigma Chemical, Type A) mixed with de-ionized water. The mixture was prepared by adding 20 ml of de-ionized water to 2 g of the biogelatin and then heated to 60 C with a magnetic stirrer until it became clear. The drug was simulated by encapsulating Direct Red by CS coated PLGA NPs, denoted as DRN. The liquid biogelatin was then poured into 10 10 30 mm cuvettes and covered by 1mm thick DRN solution to model drug on tissue. The experiments were performed using a 30 W CO2 laser (SM medical) operat-
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FULL ARTICLE J. Biophotonics 4, No. 6 (2011)
ing in continuous (CWC), single pulse (CWS), and chopped pulse repetition (PLS-R) with 10 ms pulse duration and 5 Hz.
2.4 Characterization of PLGA and PLGA/CS NPs 2.4.1 Morphology and zeta potential The hydrodynamic size of NPs in aqueous solution was determined at 25 C using laser light scattering with zeta potential measurement (Zetasizer ZS, Malvern, UK). The zeta potential of various NPs in deionized water was determined using the same analyzer. The samples were prepared by suspending the freeze-dried NPs in 5 ml deionized water. Scanning electron microscopy (SEM, Vega 2, Tescan, Chek) was employed to determine the shape and surface morphology of the produced NPs. To examine the morphology of NPs, a small amount of NPs was stuck on a double-sided tape attached on a metallic sample stand, then coated under vacuum with a thin layer of gold before SEM. The experiment was repeated three times and results were presented as means and standard deviations from the triplicate (n ¼ 3). Significance in data between different process variables was assessed using all data points obtained over multiple batches via student’s t-test and one way ANOVA with post-test. P value
