DrugRes/2014-04-0683/14.1.2015/MPS
Original Article
Authors
B. Darvishi1, 2, S. Manoochehri1, 2, M. Esfandyari-Manesh2, 3, N. Samadi4, M. Amini5, F. Atyabi1, 2, R. Dinarvand1, 2
Affiliations
Affiliation addresses are listed at the end of the article
Key words ▶ PLGA nanoparticles ● ▶ albumin conjugation ● ▶ 18-β-glycyrrhetinic acid ● ▶ toxicity ● ▶ antibacterial effect ●
Abstract
▼
The aim of the present work was to encapsulate 18-β-Glycyrrhetinic acid (GLA) in albumin conjugated poly(lactide-co-glycolide) (PLGA) nanoparticles by a modified nanoprecipitation method. Nanoparticles (NPs) were prepared by different drug to polymer ratios, human serum albumin (HSA) content, dithiothreitol (as producer of free thiol groups) content, and acetone (as non- solvent in nanoprecipitation). NPs with a size ranging from 126 to 174 nm were achieved. The highest entrapment efficiency (89.4 ± 4.2 %) was achieved when the ratio of drug to polymer was 1:4. The zeta potential of NPs was fairly negative
Introduction
▼ received 06.04.2014 accepted 02.09.2014 Bibliography DOI http://dx.doi.org/ 10.1055/s-0034-1390487 Published online: 2015 Drug Res © Georg Thieme Verlag KG Stuttgart · New York ISSN 2194-9379 Correspondence R. Dinarvand Department of Pharmaceutics Faculty of Pharmacy Tehran University of Medical Sciences Tehran 1417614411 Iran Tel.: + 98/21/66959 095 Fax: + 98/21/66959 096 [email protected]
Cancer chemotherapy is usually limited due to chemotherapeutic agents toxicity to normal tissues, short circulation half-life in plasma, nonselectivity, and limited aqueous solubility [1]. According to the fact that intracellular infections are difficult to eradicate [2] and the prevalence of their reappearance is growing up [3], studies have been extensively dedicated to the discovery of new ways and methods in order to improve treatments efficacies. One of these techniques is to deliver drugs into the specific organs using specific drug carriers. Different drug carriers like soluble polymers, polymeric NPs, liposomes, and microspheres have been investigated to increase the efficacy and decrease the side effects of anticancer and antibacterial drugs [1, 4]. Recently, nanoparticulate systems containing anticancer and antibacterial drugs have received great deal attention due to their various favorable features such as their unique ability of accumulating at the tumoric and inflamed tissues,the ability of extensive control over various properties of nanoparticle (including their size, zeta
( − 8 to − 12). Fourier transform infrared spectroscopy and differential scanning calorimetry proved the conjugation of HSA to PLGA NPs. In vitro release profile of NPs showed 2 phases: an initial burst for 4 h (34–49 %) followed by a slow release pattern up to the end. The antibacterial effects of NPs against Staphylococcus aureus, Staphylococcus epidermidis and Pseudomonas aeruginosa were studied by microdilution method. The GLA-loaded NPs showed more antibacterial effect than pure GLA (2–4 times). The anticancer MTT test revealed that GLA-loaded NPs were approximately 9 times more effective than pure GLA in Hep G2 cells.
potentials and …), stable structure, the ability of being prepared with a narrow size distribution and finally, easy surface functionalization by different groups [5]. PLGA nanoparticulate system is one of the most promising carrier system owing to its biodegradability and biocompatibility, FDA and European Medicine Agency approval, protecting the drug molecules from degradation and possibility of achieving a sustained release profile. However, one of the most important weak points of PLGA NPs is their high level of opsonization by reticuloendothelial system (RES) [6]. This deficiency was overcome by coating the surface of PLGA NPs with more hydrophilic agents such as polyethylene glycol (PEG), poloxamers and chitosan molecules to neutralize or reduce the negative zeta potential of PLGA NPs and reduce their phagocytosis by RES [7]. Among these hydrophilic compounds, human serum albumin (HSA) seems to be a new promising agent due to its passive targeting through enhanced permeation and retention (EPR) in inflamed tissues [8]. Furthermore it has been shown that HSA coated NPs can actively target tumor cells through special albumin recep-
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Enhanced Cellular Cytotoxicity and Antibacterial Activity of 18-β-Glycyrrhetinic Acid by Albuminconjugated PLGA Nanoparticles
DrugRes/2014-04-0683/14.1.2015/MPS
Original Article
Materials and Methods
▼
Materials
PLGA (50:50, 504H, MW = 48 kDa) was purchased from BoehringerIngelheim (Ingelheim, Germany). HSA, GLA, and MTT were purchased from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco’s
modified eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin and streptomycin antibiotic mixture were obtained from Life technologies (grand Island, NY, USA). N,N′-Dicyclo hexylcarbodiimide (DCC), N-hydroxysuccinimide (NHS), dithiothreitol (DTT), ethanol, acetonitrile and acetone were from Merck (Darmstadt, Germany). All other materials used were of analytical or HPLC grade.
Introduction of thiol groups on the surface of HSA
25–100 mg of unmodified HSA was dissolved in 100 mL phosphate buffer (pH=7.5). In the next step, different amounts of DTT were added to the solution and incubated for 5 h in room temperature. The solution was then extensively dialyzed with cellulose membrane (Sigma, St. Louis, MO, USA, cut-off: 12 kDa) against 2 L of 0.01 M phosphate buffer (PH=7.5) in order to remove DTT.
Preparation of para-maleimido benzoic hydrazide derivative of PLGA
Briefly 0.28 mmol PLGA, 1.1 mmol NHS and 0.15 mmol DCC were dissolved in dichloromethane and stirred for 12 h in room temperature. Then 0.42 mmol para-maleimido benzoic hydrazide (PMBH) was added to the solution and stirred for another 12 h in room temperature. The solvent was consequently evaporated by rotary evaporator. The resulting PLGA-PMBH was washed with water and desiccated for 1 week.
Preparation of PLGA-PMBH-HSA nanoparticles (PLGA-HSA NPs)
Known amounts of GLA and PLGA-PMBH were dissolved in 5 mL ▶ Table 1). The mixture was acetone at room temperature ( ● added drop wise in the rate of 2 mL/min to 100 mL phosphate buffer medium containing 100 mg modified HSA and was then stirred for 24 h. Acetone was removed at room temperature under stirring condition. The resulting suspension was centrifuged (Sigma 3K30, Germany) at 7 000 rpm for 15 min in order to remove free GLA molecules and unreacted agglomerated PLGA molecules from medium. Finally the produced suspension was freeze-dried at − 40 °C for 48 h (Christ Alpha 1–4; Germany). The final dry powder was taken out for physicochemical, cytotoxicity and antibacterial investigations.
Differential scanning calorimetry (DSC)
Thermograms of GLA, PLGA-PMBH, HSA, NPs, and physical mixture of drug with components were performed on a Mettler DSC
Table 1 PLGA nanoparticles conjugated with HSA characteristics. Sample S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 S11 S12
PLGA (mg) 5 10 20 10 10 10 10 10 10 10 10 10
HSA (mg) 50 50 50 25 75 100 50 50 50 50 50 50
DTT (mg) 5 5 5 5 5 5 1 10 15 5 5 5
O/W ratio ( %) 10 10 10 10 10 10 10 10 10 5 15 20
Size (nm)
PDI
126 ± 6 149 ± 5 174 ± 4 142.3 ± 4 152 ± 5 153.3 ± 6.5 147.4 ± 7 149.2 ± 5 150.7 ± 4 154.3 ± 6 145.3 ± 5 142.1 ± 6
0.14 ± 0.04 0.15 ± 0.02 0.15 ± 0.03 0.15 ± 0.03 0.14 ± 0.04 0.15 ± 0.03 0.16 ± 0.04 0.15 ± 0.02 0.15 ± 0.03 0.14 ± 0.05 0.14 ± 0.03 0.15 ± 0.04
Zeta potential (mV) − 9.9 ± 1.1 − 10.5 ± 1.3 − 12 ± 1.4 − 12.5 ± 1.3 − 10 ± 1.2 − 8.7 ± 1.1 − 9.6 ± 1.2 − 9.4 ± 1.1 − 8.9 ± 1.2 − 8.9 ± 1.3 − 9.4 ± 1.2 − 9.5 ± 1.2
E.E ( %) 64 ± 4 73 ± 5 89.4 ± 3 68 ± 4 74 ± 4 75.3 ± 5 67.1 ± 6 69.4 ± 5 68.6 ± 2 69.4 ± 4 70.3 ± 3 72.3 ± 5
Loading ( %) 14.36 ± 1.3 10.26 ± 1.2 8.8 ± 0.9 16.8 ± 1.5 9.9 ± 0.8 6.5 ± 0.6 10.5 ± 1.2 9.9 ± 1 10.9 ± 1.1 10.3 ± 1.2 10.3 ± 1.1 10.5 ± 1.3
DTT: ditiothreithol, O: oil phase, W: water phase, HSA: human serum albumin, PLGA: Poly (lactic-co-glycolic acid), PDI: polydispersity index, and EE: entrapment efficiency
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tors on their membranes (glycoprotein receptor, gp60) [9]. HSA is the most abundant native protein in human body which has several advantages including biodegradability, low toxicity and low immunogenicity [10]. 18-β-Glycyrrhetinic acid (GLA) is a pentacyclic triterpenoid derivative of the beta-amyrin type. A great amount of evidence indicates that GLA reveals anticancer and antibacterial effects [11]. In the present work, the enhancement of anticancer and antibacterial effects of GLA was achieved by its encapsulation, in PLGA-HSA NPs. Conjugation of HSA with PLGA NPs was done through disulfide bonds between the para-maleimido benzoic hydrazide derivative of PLGA and the free –SH groups presented in HSA. The effect of several variables on the physicochemical characteristics of NPs including the amount of drug to polymer ratio, HSA content, dithiothreitol (as producer of free thiol groups) content, and acetone (as non-solvent in nanoprecipitation) were also evaluated. The encapsulation efficiency (EE) and in-vitro release of GLA from NPs were then investigated. Cytotoxicity of GLAloaded NPs was investigated by 3-(4,5-dimethyathiazol-2-yl)2,5-diphenyltetrazoliumbromide (MTT) assay in HepG2 cell line. Furthermore, the impact of nanoencapsulation of GLA on its antibacterial activity against Staphylococcus aureus (S. aureus), Staphy lococcus epidermidis (S. epidermidis), and Pseudomona aeruginosa was evaluated. These bacteria are the three most important pathogens which are considered to be involved in nosocomial infections and are internalized in phagocytic cells. S. aureus is a widespread pathogen which can also cause food poisoning [12]. S. epidermidis is another pathogen which is involved in extraneous devices and implants related infections because of its considerable potential of growing biofilms on polymer surfaces [13]. Finally Pseudomonas aeruginosa is the second ranking gram-negative bacterium causing nosocomial infection and unfortunately only limited antibacterial agents are effective against it [14]. Literature also demonstrates effectiveness of pure GLA against these bacteria species [15]. Herein we prepared GLA loaded PLGA-HSA nanoparticles in order to see if the nano form can lower the MIC or not.
DrugRes/2014-04-0683/14.1.2015/MPS
Fourier-transform infrared spectroscopy (FT-IR)
Fourier-transform infrared spectroscopy of pure GLA, PLGAPMBH, HSA and NPs were obtained on a Bomem 2000 FT-IR system (Bomem, Quebec, Canada) using the KBr disk method.
Nanoparticle size, morphology and zeta potential
For measuring the size and poly dispersity index (PDI) of NPs, lyophilized powder of NPs were dissolved in water and were measured by Photon Correlation Spectroscopy (PCS) using a Zetasizer nano ZS (Malvern Instruments, Worcestershire, UK) at 25 °C. Zeta potential of NPs was also measured by the same instrument. All the measurements were triplicate and mean ± SD was reported. The surface morphology and the shape of NPs were investigated by scanning electron microscopy (SEM, Philips XL 30, Philips, Netherlands). Samples were prepared on aluminum stubs and coated with gold under argon atmosphere by means of a sputter coater.
Encapsulation efficiency
1 mL acetonitrile was added to 5 mg of lyophilized NPs and was then sonicated for 15 min. In the next step 2 mL of methanol was added in order to precipitate the polymer. Thereafter the sample was centrifuged at 21 000 rpm for 20 min and the supernatant was separated for further analysis. Drug quantity in the supernatant was determined by UV-spectrophotometer (Scinco S-3100, Korea) at wavelength of 249 nm. Drug loading was determined as the ratio of drug content of NPs to the total weight of NPs. The encapsulation efficiency was determined as the mass ratio of entrapped GLA in NPs to the theoretical amount of GLA used in their preparation [16, 17]. The experiments were repeated 3 times.
In-vitro drug release
Drug release was performed according to the previously published method for Piroxicam loaded NPs [18] with some modification. 5 mg lyophilized NPs were dissolved in 10 mL of phosphate buffer saline (PBS, pH=7.4) and were placed in a dialysis membrane bag with a molecular weight cut-off of 12 kDa. The dialysis bag was then tied and was immersed into 50 mL of PBS medium. The entire system was kept at 37 °C in a shaker (250 rpm). After a predetermined period, 5 mL of the medium was removed and the amount of GLA was analyzed by UV-spectrophotometer. The medium was fully removed and fresh medium was replaced each time. Furthermore, the release of GLA from the dialysis bag under the same conditions was also evaluated.
In-vitro cytotoxicity
HepG2 (Human hepatocellular carcinoma) cells were obtained from Pasteur Institute of Iran (Tehran, Iran) and were cultivated and maintained in DMEM growth media supplemented with 10 % FBS and 1 % penicillin streptomycin antibiotic mixture. Cells were then incubated at 37 °C in a humidified atmosphere with 5 % CO2 exchanging the culture media every other day. Cells were seeded in 96 wells with a density of 1 × 104 cells per well and
were incubated for 24 h to allow cell attachment. Cells were then treated with different concentrations of free GLA and GLAloaded NPs for 96 h and were then exposed to 20 μl of MTT reagent (5 mg/mL) in each well for an additional 4 h. The culture media was then discarded and 200 μl dimethylsulfoxide (DMSO) was added to each well in order to dissolve the formed formazan crystals. Optical density was then measured at 570 nm with a 660 nm background using a microplate reader (Anthos 2020; Anthos Lab tec Instruments, Wals, Austria). Each experiment was performed in triplicate and cellular viability was expressed as the percentage of viable cells to control. IC50 was calculated from the cell viability data as the drug concentration which can inhibit 50% of cellular growth [19].
Antibacterial activity
Minimum inhibitory concentration (MIC) of free GLA, GLAloaded NPs, and blank NPs was determined against S. aureus ATCC 6 538, S. epidermidis ATCC 12 228, and P. aeruginosa ATCC 9 027. The antibacterial activity test was determined by microdilution method using 96 U-shaped wells plates (NCCLS, 2006). In order to verify the accuracy of results and rejecting the possibility of bacterial resistance, one control organism was included with each group of MIC determinations. The routinely used antibiotics MICs were within the published ranges of control organisms and consequently no antibacterial resistance was detected. Aliquot of 100 μl DMSO was used to dissolve 3 mg of test compound in 900 μl Mueller-Hinton broth (MHB). A 200 μL aliquot of stock solution of test compound was transferred into the first well in each row and serially diluted by mixing with 100 mL of MHB in subsequent wells. Then, 100 μL of a diluted 0.5 McFarland suspension was added to the wells in each horizontal row to reach the final inoculum size of about 5 × 105 CFU/mL. After 24 h incubation at 37 °C, the micro dilution trays were tested for the absence or presence of any visible growth. The endpoint MIC was determined as the lowest concentration of drug at which the test strain does not demonstrate visible growth.
Results and Discussion
▼
Preparation of NPs and their physicochemical properties
PLGA NPs were prepared by nanoprecipitation method with a slight modification. Nanoprecipitation occurs by a rapid desolvation of the polymer when the solvent diffuses toward the nonsolvent. HSA conjugation to PLGA NPs was done through the disulfide bond between the maleimide group of PMBH derivative of PLGA and the free sulfide groups of HSA. Before conjugation, the thiol groups in HSA were introduced in its structure by reacting with DTT. In order to obtain optimal formulation (particle size and drug encapsulation efficiency), several parameters were evaluated including PLGA content, HSA content, DTT content, and volume ▶ Table 1). ratio of organic to aqueous phase ( ● ▶ Fig. 1a) showed that PLGA-HSA NPs formulated SEM results ( ● with 5 mg PLGA have smooth surface and spherical shape without any agglomerated particles. However NPs formulated with 20 mg PLGA content showed both agglomerated and non-spher▶ Fig. 1b).This phenomenon was also observed by ical particles ( ● Mainardes et al. for PLGA nanoparticles containing praziquantel [20]. The agglomerated and non-spherical particles were disap▶ Fig. 1c). peared by increasing of the HSA to PLGA ratio ( ● Darvishi B et al. Glycyrrhetinic Acid Loaded Nanoparticles … Drug Res
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823 (Mettler Toledo, GmbH, Switzerland) equipped with Mettler STARe system software for data acquisition. Samples were placed in aluminum pans and the lids were crimped using a crimper. Samples were scanned at a scanning rate of 5 °C/min covering temperature range of 25–350 °C. The instrument was calibrated with an indium standard.
Original Article
DrugRes/2014-04-0683/14.1.2015/MPS
Original Article
a
b
Fig. 1 Scanning electron microscopy of a Nanoparticles prepared with 5 mg PLGA-PMBH and 25 mg HSA, b Nanoparticles prepared with 20 mg PLGA-PMBH and 25 mg HSA, c Nanoparticles prepared with 20 mg PLGA and 50 mg of HSA.
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c
PLGA content ▶ Table 1 shows the influence of PLGA content variation on the ●
size, PDI and encapsulation efficiency of NPs. By increasing the PLGA content from 5 to 20 mg the particle diameter increased from 126 to 174 nm. Increase in the size of NPs may be related to the higher viscosity of polymer in the injection phase. Higher encapsulation efficiencies were obtained by increasing the amount of polymer. Maximum encapsulation efficiency was achieved when the amount of polymer was 20 mg.
HSA content As shown in ● ▶ Table 1 by increasing the amount of HSA from 25
to 100 mg, the particle diameter was increased slightly from 142.3 to 153.3 nm. Increasing HSA content results in more HSA conjugation to PLGA NPs. However there is a border line in this phenomenon. The increasing of HSA content from 75 to 100 mg causes no significant increase in nanoparticles diameter. This can be explained by special hindrance exposed by HSA molecules.
DTT content
No significant changes appeared in nanoparticles size by increasing the amount of DTT. It can be concluded that small amounts of DTT can activate enough HSA molecules for reacting with thiol groups in PLGA-PMBH polymers.
Organic to aqueous phase volume ratio
The ratio between internal and external phases showed a small effect on size of NPs. By increasing the organic to aqueous phase percent from 5 to 20 %, a slight decrease appeared in size of NPs. Low polymer viscosity produces smaller organic droplets. The coalescence of organic droplets can be prevented by a large amount of organic solvent available for diffusion [20].
Differential scanning calorimetry (DSC) The DSC results in ● ▶ Fig. 2 provide quantitative information
about the physical state of the drug in NPs. Pure PLGA exhibited an endothermic peak at 60 °C which refers to the glass transition temperature of polymer. No melting point was detected, because
Darvishi B et al. Glycyrrhetinic Acid Loaded Nanoparticles … Drug Res
Fig. 2 DSC thermograms of PLGA, HSA, GLA and GLA loaded PLGA-HSA NPs.
PLGA appears amorphous in nature [21]. Pure HSA shows an endothermic peak at 70 °C which is due to its melting point [22]. Pure GLA had an endothermic peak at 300 °C corresponding to its melting point. The DSC curve of GLA, HSA and PLGA in their physical mixture shows the peaks resulting from overlapping of the DSC curves of the separated components. Thus, no interaction and crystalline change could be attributed to the heating process. The DSC curve of GLA-loaded NPs shows the event corresponding to the glass transition temperature of PLGA (60 °C) and also the peak in correspondence to the melting point of albumin (70 °C) which confirms the existence of albumin in the structure of NPs. In GLA-loaded NPs, the endothermic peak of GLA was absent. DSC studies did not detect any free GLA in the GLA-loaded NPs sample. Thus, it can be concluded that the drug incorporated into NPs was in an amorphous state in the polymer
DrugRes/2014-04-0683/14.1.2015/MPS
Original Article
In-vitro release studies
Fig. 4 GLA release pattern from S1 and S2 in PBS medium (pH = 7.4).
matrix. This confirmed the molecular dispersion of GLA in the polymer matrix.
Fourier-transform infrared spectroscopy
FT-IR spectra of PLGA, HSA and HSA conjugated PLGA NPs are shown in ● ▶ Fig. 3. HSA spectrum shows characteristic peaks at 3 430, 3 062, 1 652, and 1 531 cm − 1 which are assigned to the stretching vibration of –OH, amide A (mainly –NH stretching vibration), amide (mainly –C = O stretching vibrations), and amide (the coupling of bending vibrate of N–H and stretching vibrate of C–N) bands, respectively. Pure PLGA sample shows peaks such as –CH, –CH2, –CH3 stretching (2 850–3 000 cm − 1), carbonyl –C = O stretching (1 747 cm − 1), C–O stretching (1 050–1 250 cm − 1), and –OH stretching (3 200–3 500 cm − 1). When HSA conjugated PLGA NPs spectrum was evaluated, 1 750 cm − 1 (carbonyl –C = O stretching of ester) and 1 643 cm − 1 (carbonyl –C = O stretching of amide) absorption peaks were observed which confirms the presence of both PLGA and HSA components in the conjugated NPs [23].
The in-vitro release profile of the pure GLA and GLA-loaded NPs with different PLGA contents are shown in ● ▶ Fig. 4. The time needed for dissolving 50 % of drug (t50 %) which is inversely related to the release rate, are: 3 h for pure GLA, 19.5 h for GLAloaded NPs prepared by 5 mg PLGA, and 67 h for GLA-loaded NPs prepared by 20 mg PLGA. It is evident that an increase in the amount of polymer in the nanoparticles would cause a more reduction in release rate of GLA from NPs. ▶ Fig. 4 shows that the release of GLA from NPs was slower and ● more sustained than that of pure GLA. The rate of drug release from any solid or semi-solid delivery system is usually controlled by dissolution and/or diffusion [24]. Here in, the release profiles of GLA from nanoparticle formulations could be divided in 2 phases: an initial burst release for 4 h followed by a very slow release pattern for the rest of release profile. The quick first release of GLA is ascribed to the dissolution and diffusion of the drug that was poorly entrapped in the polymer matrix, while in the longer time periods the GLA molecules that are more tightly entrapped to the polymer matrix are released. The amount of initial burst release depends upon the amount of PLGA in the nanoparticle samples. The results showed that during the first 4 h, an initial burst release led to an early release of 49.8 and 34.2 % of drug from NPs with 5 and 10 mg PLGA, respectively.
In vitro cytotoxicity studies
HepG2 cell line was used in order to assess the in vitro cellular cytotoxicity of the samples. HepG2 cell line is considered as one of the suitable models for cytotoxicity studies [25]. Following 72 h of exposure to the GLA-loaded NPs and free GLA, cell viabil▶ Fig. 5). The cytotoxicity results ity was assessed by MTT assay ( ● are presented in ● ▶ Table 2. The IC50 of free GLA, and GLA-loaded NPs were 83.4 ± 0.56 and 9.2 ± 0.21 µg/mL respectively which is approximately 9 times more toxic after 72 h period of incubation in the cell line. The result indicates that the GLA formulated in the PLGA-HSA NPs has shown advantages in achieving lower cell viability, and or equivalently higher cytotoxicity, vs. free GLA. When GLA is
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Fig. 3 FT-IR spectrum of HSA, PLGA, their physical mixture (HSA and PLGA) and PLGA NPs conjugated with HSA.
DrugRes/2014-04-0683/14.1.2015/MPS
Original Article
has been revealed that NPs are able to be endocytosed by phagocytic cells and release the drug molecules inside the cells [19]. GLA-loaded NPs could be useful in drug targeting to the phagocytic cells to improve the treatment of intracellular infections compared to the treatments using free antibiotics. Other NPs have been evaluated for ocular [28] and oral [29] administration, which suggests that the formulated GLA-loaded NPs could be useful for the mentioned routes of administration.
Conclusion
Table 2 IC50 of GLA, GLA loaded NPs and drug free NPs against Hep-G2 cell line. Sample
IC50 (µg/l)
GLA GLA-loaded PLGA-HSA NPs Drug free NPs
83.4 ± 0.56 9.2 ± 0.21 –
Table 3 Minimum inhibitory concentrations (μg/l) of GLA, GLA loaded NPs and drug free NPs against selected bacteria. Sample GLA GLA-loaded PLGA-HSA NPs Drug free NPs
S. aureus 150 75 –
S. epidermidis 37 9.3 –
P. aeruginosa 75 18.7 –
In conclusion, an albumin-conjugated PLGA NPs of GLA was developed that had higher anticancer and antibacterial activity than free drug. HSA conjugation to PLGA NPs was achieved through disulfide bond formation between the para-maleimido benzoic hydrazide derivative of PLGA and HSA. Different nanoparticles with various drug to polymer ratio, HSA content, DTT content, and acetone volume were evaluated. The drug to polymer ratio had significant effect on the size and morphology, the drug loading, and release profile. It was shown that GLA-loaded PLGA-HSA NPs, provided desirable drug loading and size characteristics making them suitable for i. v. administration. The developed nanoparticles presented more than 9 times cellular mortality compared to free GLA. The release studies indicated that after the initial burst release, controlled release of GLA continued for more than 6 days; this release profile could be the ideal for antibacterial application.
Conflict of Interest
▼
The authors declare no conflict of interest. encapsulated in PLGA-HSA NPs, a higher GLA concentration is transported to the intracellular space which may be due to the small size of NPs and the passive targeting of macromolecules resulted from EPR effect. As a result a better efficacy and a lower IC50 is observed for PLGA NPs conjugated with HSA [21]. The outer layer of PLGA-HSA NPs, forms a stealth surface with a threshold gap that can reduce the NPs interaction (both between NPs and between cells and NPs) [22, 26]. Similar MTT assay was performed with PLGA-HSA NPs without encapsulating GLA to assess any intrinsic toxicity. GLA-unloaded NPs did not show any cytotoxic effect (data not shown).
Antimicrobial activity of the nanoparticle suspensions
The MIC of GLA-loaded NPs and free GLA on S. aureus, S. epider▶ Table 3). GLAmidis, and Ps. aeroginosa are presented in ( ● unloaded NPs showed no antibacterial activity which indicates that none of the ingredients of formulation have any antibacterial effect. The MIC of GLA-loaded NPs were approximately 2 times lower on S. aureus, 3 times lower on S. epidermidis, and 4 times lower on Ps. aeruginosa compared to free GLA. This indicates that the effective dose of this antibiotic can be reduced against the above bacteria and hence the side effects of the drug will reduce. Consequently, in vitro anti-microbial activity of GLA-loaded NPs was better than the free drug. In other studies enhanced antimicrobial activity from PLGA NPs containing antimicrobial agents has been reported [19, 27]. This may be related to the improved penetration of NPs from biological membranes. For GLA to be effective it should pass through the bacterial membrane in order to inhibit the synthesis of DNA and RNA. It Darvishi B et al. Glycyrrhetinic Acid Loaded Nanoparticles … Drug Res
Affiliations 1 Department of Pharmaceutics, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran 2 Nanotechnology Research Center, Tehran University of Medical Sciences, Tehran, Iran 3 Department of Chemistry, Amirkabir University of Technology, Tehran, Iran 4 Drug and Food Control Department, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran 5 Department of Medicinal Chemistry, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran
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▼ Fig. 5 Cell viability in HepG2 cell line in exposure to free GLA and GLA encapsulated PLGA-HSA with various concentrations against Hep-G2 assessed by MTT assay (n = 3).
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DrugRes/2014-04-0683/14.1.2015/MPS
Original Article
Authors
B. Darvishi1, 2, S. Manoochehri1, 2, M. Esfandyari-Manesh2, 3, N. Samadi4, M. Amini5, F. Atyabi1, 2, R. Dinarvand1, 2
Affiliations
Affiliation addresses are listed at the end of the article
Key words ▶ PLGA nanoparticles ● ▶ albumin conjugation ● ▶ 18-β-glycyrrhetinic acid ● ▶ toxicity ● ▶ antibacterial effect ●
Abstract
▼
The aim of the present work was to encapsulate 18-β-Glycyrrhetinic acid (GLA) in albumin conjugated poly(lactide-co-glycolide) (PLGA) nanoparticles by a modified nanoprecipitation method. Nanoparticles (NPs) were prepared by different drug to polymer ratios, human serum albumin (HSA) content, dithiothreitol (as producer of free thiol groups) content, and acetone (as non- solvent in nanoprecipitation). NPs with a size ranging from 126 to 174 nm were achieved. The highest entrapment efficiency (89.4 ± 4.2 %) was achieved when the ratio of drug to polymer was 1:4. The zeta potential of NPs was fairly negative
Introduction
▼ received 06.04.2014 accepted 02.09.2014 Bibliography DOI http://dx.doi.org/ 10.1055/s-0034-1390487 Published online: 2015 Drug Res © Georg Thieme Verlag KG Stuttgart · New York ISSN 2194-9379 Correspondence R. Dinarvand Department of Pharmaceutics Faculty of Pharmacy Tehran University of Medical Sciences Tehran 1417614411 Iran Tel.: + 98/21/66959 095 Fax: + 98/21/66959 096 [email protected]
Cancer chemotherapy is usually limited due to chemotherapeutic agents toxicity to normal tissues, short circulation half-life in plasma, nonselectivity, and limited aqueous solubility [1]. According to the fact that intracellular infections are difficult to eradicate [2] and the prevalence of their reappearance is growing up [3], studies have been extensively dedicated to the discovery of new ways and methods in order to improve treatments efficacies. One of these techniques is to deliver drugs into the specific organs using specific drug carriers. Different drug carriers like soluble polymers, polymeric NPs, liposomes, and microspheres have been investigated to increase the efficacy and decrease the side effects of anticancer and antibacterial drugs [1, 4]. Recently, nanoparticulate systems containing anticancer and antibacterial drugs have received great deal attention due to their various favorable features such as their unique ability of accumulating at the tumoric and inflamed tissues,the ability of extensive control over various properties of nanoparticle (including their size, zeta
( − 8 to − 12). Fourier transform infrared spectroscopy and differential scanning calorimetry proved the conjugation of HSA to PLGA NPs. In vitro release profile of NPs showed 2 phases: an initial burst for 4 h (34–49 %) followed by a slow release pattern up to the end. The antibacterial effects of NPs against Staphylococcus aureus, Staphylococcus epidermidis and Pseudomonas aeruginosa were studied by microdilution method. The GLA-loaded NPs showed more antibacterial effect than pure GLA (2–4 times). The anticancer MTT test revealed that GLA-loaded NPs were approximately 9 times more effective than pure GLA in Hep G2 cells.
potentials and …), stable structure, the ability of being prepared with a narrow size distribution and finally, easy surface functionalization by different groups [5]. PLGA nanoparticulate system is one of the most promising carrier system owing to its biodegradability and biocompatibility, FDA and European Medicine Agency approval, protecting the drug molecules from degradation and possibility of achieving a sustained release profile. However, one of the most important weak points of PLGA NPs is their high level of opsonization by reticuloendothelial system (RES) [6]. This deficiency was overcome by coating the surface of PLGA NPs with more hydrophilic agents such as polyethylene glycol (PEG), poloxamers and chitosan molecules to neutralize or reduce the negative zeta potential of PLGA NPs and reduce their phagocytosis by RES [7]. Among these hydrophilic compounds, human serum albumin (HSA) seems to be a new promising agent due to its passive targeting through enhanced permeation and retention (EPR) in inflamed tissues [8]. Furthermore it has been shown that HSA coated NPs can actively target tumor cells through special albumin recep-
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Enhanced Cellular Cytotoxicity and Antibacterial Activity of 18-β-Glycyrrhetinic Acid by Albuminconjugated PLGA Nanoparticles
DrugRes/2014-04-0683/14.1.2015/MPS
Original Article
Materials and Methods
▼
Materials
PLGA (50:50, 504H, MW = 48 kDa) was purchased from BoehringerIngelheim (Ingelheim, Germany). HSA, GLA, and MTT were purchased from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco’s
modified eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin and streptomycin antibiotic mixture were obtained from Life technologies (grand Island, NY, USA). N,N′-Dicyclo hexylcarbodiimide (DCC), N-hydroxysuccinimide (NHS), dithiothreitol (DTT), ethanol, acetonitrile and acetone were from Merck (Darmstadt, Germany). All other materials used were of analytical or HPLC grade.
Introduction of thiol groups on the surface of HSA
25–100 mg of unmodified HSA was dissolved in 100 mL phosphate buffer (pH=7.5). In the next step, different amounts of DTT were added to the solution and incubated for 5 h in room temperature. The solution was then extensively dialyzed with cellulose membrane (Sigma, St. Louis, MO, USA, cut-off: 12 kDa) against 2 L of 0.01 M phosphate buffer (PH=7.5) in order to remove DTT.
Preparation of para-maleimido benzoic hydrazide derivative of PLGA
Briefly 0.28 mmol PLGA, 1.1 mmol NHS and 0.15 mmol DCC were dissolved in dichloromethane and stirred for 12 h in room temperature. Then 0.42 mmol para-maleimido benzoic hydrazide (PMBH) was added to the solution and stirred for another 12 h in room temperature. The solvent was consequently evaporated by rotary evaporator. The resulting PLGA-PMBH was washed with water and desiccated for 1 week.
Preparation of PLGA-PMBH-HSA nanoparticles (PLGA-HSA NPs)
Known amounts of GLA and PLGA-PMBH were dissolved in 5 mL ▶ Table 1). The mixture was acetone at room temperature ( ● added drop wise in the rate of 2 mL/min to 100 mL phosphate buffer medium containing 100 mg modified HSA and was then stirred for 24 h. Acetone was removed at room temperature under stirring condition. The resulting suspension was centrifuged (Sigma 3K30, Germany) at 7 000 rpm for 15 min in order to remove free GLA molecules and unreacted agglomerated PLGA molecules from medium. Finally the produced suspension was freeze-dried at − 40 °C for 48 h (Christ Alpha 1–4; Germany). The final dry powder was taken out for physicochemical, cytotoxicity and antibacterial investigations.
Differential scanning calorimetry (DSC)
Thermograms of GLA, PLGA-PMBH, HSA, NPs, and physical mixture of drug with components were performed on a Mettler DSC
Table 1 PLGA nanoparticles conjugated with HSA characteristics. Sample S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 S11 S12
PLGA (mg) 5 10 20 10 10 10 10 10 10 10 10 10
HSA (mg) 50 50 50 25 75 100 50 50 50 50 50 50
DTT (mg) 5 5 5 5 5 5 1 10 15 5 5 5
O/W ratio ( %) 10 10 10 10 10 10 10 10 10 5 15 20
Size (nm)
PDI
126 ± 6 149 ± 5 174 ± 4 142.3 ± 4 152 ± 5 153.3 ± 6.5 147.4 ± 7 149.2 ± 5 150.7 ± 4 154.3 ± 6 145.3 ± 5 142.1 ± 6
0.14 ± 0.04 0.15 ± 0.02 0.15 ± 0.03 0.15 ± 0.03 0.14 ± 0.04 0.15 ± 0.03 0.16 ± 0.04 0.15 ± 0.02 0.15 ± 0.03 0.14 ± 0.05 0.14 ± 0.03 0.15 ± 0.04
Zeta potential (mV) − 9.9 ± 1.1 − 10.5 ± 1.3 − 12 ± 1.4 − 12.5 ± 1.3 − 10 ± 1.2 − 8.7 ± 1.1 − 9.6 ± 1.2 − 9.4 ± 1.1 − 8.9 ± 1.2 − 8.9 ± 1.3 − 9.4 ± 1.2 − 9.5 ± 1.2
E.E ( %) 64 ± 4 73 ± 5 89.4 ± 3 68 ± 4 74 ± 4 75.3 ± 5 67.1 ± 6 69.4 ± 5 68.6 ± 2 69.4 ± 4 70.3 ± 3 72.3 ± 5
Loading ( %) 14.36 ± 1.3 10.26 ± 1.2 8.8 ± 0.9 16.8 ± 1.5 9.9 ± 0.8 6.5 ± 0.6 10.5 ± 1.2 9.9 ± 1 10.9 ± 1.1 10.3 ± 1.2 10.3 ± 1.1 10.5 ± 1.3
DTT: ditiothreithol, O: oil phase, W: water phase, HSA: human serum albumin, PLGA: Poly (lactic-co-glycolic acid), PDI: polydispersity index, and EE: entrapment efficiency
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tors on their membranes (glycoprotein receptor, gp60) [9]. HSA is the most abundant native protein in human body which has several advantages including biodegradability, low toxicity and low immunogenicity [10]. 18-β-Glycyrrhetinic acid (GLA) is a pentacyclic triterpenoid derivative of the beta-amyrin type. A great amount of evidence indicates that GLA reveals anticancer and antibacterial effects [11]. In the present work, the enhancement of anticancer and antibacterial effects of GLA was achieved by its encapsulation, in PLGA-HSA NPs. Conjugation of HSA with PLGA NPs was done through disulfide bonds between the para-maleimido benzoic hydrazide derivative of PLGA and the free –SH groups presented in HSA. The effect of several variables on the physicochemical characteristics of NPs including the amount of drug to polymer ratio, HSA content, dithiothreitol (as producer of free thiol groups) content, and acetone (as non-solvent in nanoprecipitation) were also evaluated. The encapsulation efficiency (EE) and in-vitro release of GLA from NPs were then investigated. Cytotoxicity of GLAloaded NPs was investigated by 3-(4,5-dimethyathiazol-2-yl)2,5-diphenyltetrazoliumbromide (MTT) assay in HepG2 cell line. Furthermore, the impact of nanoencapsulation of GLA on its antibacterial activity against Staphylococcus aureus (S. aureus), Staphy lococcus epidermidis (S. epidermidis), and Pseudomona aeruginosa was evaluated. These bacteria are the three most important pathogens which are considered to be involved in nosocomial infections and are internalized in phagocytic cells. S. aureus is a widespread pathogen which can also cause food poisoning [12]. S. epidermidis is another pathogen which is involved in extraneous devices and implants related infections because of its considerable potential of growing biofilms on polymer surfaces [13]. Finally Pseudomonas aeruginosa is the second ranking gram-negative bacterium causing nosocomial infection and unfortunately only limited antibacterial agents are effective against it [14]. Literature also demonstrates effectiveness of pure GLA against these bacteria species [15]. Herein we prepared GLA loaded PLGA-HSA nanoparticles in order to see if the nano form can lower the MIC or not.
DrugRes/2014-04-0683/14.1.2015/MPS
Fourier-transform infrared spectroscopy (FT-IR)
Fourier-transform infrared spectroscopy of pure GLA, PLGAPMBH, HSA and NPs were obtained on a Bomem 2000 FT-IR system (Bomem, Quebec, Canada) using the KBr disk method.
Nanoparticle size, morphology and zeta potential
For measuring the size and poly dispersity index (PDI) of NPs, lyophilized powder of NPs were dissolved in water and were measured by Photon Correlation Spectroscopy (PCS) using a Zetasizer nano ZS (Malvern Instruments, Worcestershire, UK) at 25 °C. Zeta potential of NPs was also measured by the same instrument. All the measurements were triplicate and mean ± SD was reported. The surface morphology and the shape of NPs were investigated by scanning electron microscopy (SEM, Philips XL 30, Philips, Netherlands). Samples were prepared on aluminum stubs and coated with gold under argon atmosphere by means of a sputter coater.
Encapsulation efficiency
1 mL acetonitrile was added to 5 mg of lyophilized NPs and was then sonicated for 15 min. In the next step 2 mL of methanol was added in order to precipitate the polymer. Thereafter the sample was centrifuged at 21 000 rpm for 20 min and the supernatant was separated for further analysis. Drug quantity in the supernatant was determined by UV-spectrophotometer (Scinco S-3100, Korea) at wavelength of 249 nm. Drug loading was determined as the ratio of drug content of NPs to the total weight of NPs. The encapsulation efficiency was determined as the mass ratio of entrapped GLA in NPs to the theoretical amount of GLA used in their preparation [16, 17]. The experiments were repeated 3 times.
In-vitro drug release
Drug release was performed according to the previously published method for Piroxicam loaded NPs [18] with some modification. 5 mg lyophilized NPs were dissolved in 10 mL of phosphate buffer saline (PBS, pH=7.4) and were placed in a dialysis membrane bag with a molecular weight cut-off of 12 kDa. The dialysis bag was then tied and was immersed into 50 mL of PBS medium. The entire system was kept at 37 °C in a shaker (250 rpm). After a predetermined period, 5 mL of the medium was removed and the amount of GLA was analyzed by UV-spectrophotometer. The medium was fully removed and fresh medium was replaced each time. Furthermore, the release of GLA from the dialysis bag under the same conditions was also evaluated.
In-vitro cytotoxicity
HepG2 (Human hepatocellular carcinoma) cells were obtained from Pasteur Institute of Iran (Tehran, Iran) and were cultivated and maintained in DMEM growth media supplemented with 10 % FBS and 1 % penicillin streptomycin antibiotic mixture. Cells were then incubated at 37 °C in a humidified atmosphere with 5 % CO2 exchanging the culture media every other day. Cells were seeded in 96 wells with a density of 1 × 104 cells per well and
were incubated for 24 h to allow cell attachment. Cells were then treated with different concentrations of free GLA and GLAloaded NPs for 96 h and were then exposed to 20 μl of MTT reagent (5 mg/mL) in each well for an additional 4 h. The culture media was then discarded and 200 μl dimethylsulfoxide (DMSO) was added to each well in order to dissolve the formed formazan crystals. Optical density was then measured at 570 nm with a 660 nm background using a microplate reader (Anthos 2020; Anthos Lab tec Instruments, Wals, Austria). Each experiment was performed in triplicate and cellular viability was expressed as the percentage of viable cells to control. IC50 was calculated from the cell viability data as the drug concentration which can inhibit 50% of cellular growth [19].
Antibacterial activity
Minimum inhibitory concentration (MIC) of free GLA, GLAloaded NPs, and blank NPs was determined against S. aureus ATCC 6 538, S. epidermidis ATCC 12 228, and P. aeruginosa ATCC 9 027. The antibacterial activity test was determined by microdilution method using 96 U-shaped wells plates (NCCLS, 2006). In order to verify the accuracy of results and rejecting the possibility of bacterial resistance, one control organism was included with each group of MIC determinations. The routinely used antibiotics MICs were within the published ranges of control organisms and consequently no antibacterial resistance was detected. Aliquot of 100 μl DMSO was used to dissolve 3 mg of test compound in 900 μl Mueller-Hinton broth (MHB). A 200 μL aliquot of stock solution of test compound was transferred into the first well in each row and serially diluted by mixing with 100 mL of MHB in subsequent wells. Then, 100 μL of a diluted 0.5 McFarland suspension was added to the wells in each horizontal row to reach the final inoculum size of about 5 × 105 CFU/mL. After 24 h incubation at 37 °C, the micro dilution trays were tested for the absence or presence of any visible growth. The endpoint MIC was determined as the lowest concentration of drug at which the test strain does not demonstrate visible growth.
Results and Discussion
▼
Preparation of NPs and their physicochemical properties
PLGA NPs were prepared by nanoprecipitation method with a slight modification. Nanoprecipitation occurs by a rapid desolvation of the polymer when the solvent diffuses toward the nonsolvent. HSA conjugation to PLGA NPs was done through the disulfide bond between the maleimide group of PMBH derivative of PLGA and the free sulfide groups of HSA. Before conjugation, the thiol groups in HSA were introduced in its structure by reacting with DTT. In order to obtain optimal formulation (particle size and drug encapsulation efficiency), several parameters were evaluated including PLGA content, HSA content, DTT content, and volume ▶ Table 1). ratio of organic to aqueous phase ( ● ▶ Fig. 1a) showed that PLGA-HSA NPs formulated SEM results ( ● with 5 mg PLGA have smooth surface and spherical shape without any agglomerated particles. However NPs formulated with 20 mg PLGA content showed both agglomerated and non-spher▶ Fig. 1b).This phenomenon was also observed by ical particles ( ● Mainardes et al. for PLGA nanoparticles containing praziquantel [20]. The agglomerated and non-spherical particles were disap▶ Fig. 1c). peared by increasing of the HSA to PLGA ratio ( ● Darvishi B et al. Glycyrrhetinic Acid Loaded Nanoparticles … Drug Res
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823 (Mettler Toledo, GmbH, Switzerland) equipped with Mettler STARe system software for data acquisition. Samples were placed in aluminum pans and the lids were crimped using a crimper. Samples were scanned at a scanning rate of 5 °C/min covering temperature range of 25–350 °C. The instrument was calibrated with an indium standard.
Original Article
DrugRes/2014-04-0683/14.1.2015/MPS
Original Article
a
b
Fig. 1 Scanning electron microscopy of a Nanoparticles prepared with 5 mg PLGA-PMBH and 25 mg HSA, b Nanoparticles prepared with 20 mg PLGA-PMBH and 25 mg HSA, c Nanoparticles prepared with 20 mg PLGA and 50 mg of HSA.
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c
PLGA content ▶ Table 1 shows the influence of PLGA content variation on the ●
size, PDI and encapsulation efficiency of NPs. By increasing the PLGA content from 5 to 20 mg the particle diameter increased from 126 to 174 nm. Increase in the size of NPs may be related to the higher viscosity of polymer in the injection phase. Higher encapsulation efficiencies were obtained by increasing the amount of polymer. Maximum encapsulation efficiency was achieved when the amount of polymer was 20 mg.
HSA content As shown in ● ▶ Table 1 by increasing the amount of HSA from 25
to 100 mg, the particle diameter was increased slightly from 142.3 to 153.3 nm. Increasing HSA content results in more HSA conjugation to PLGA NPs. However there is a border line in this phenomenon. The increasing of HSA content from 75 to 100 mg causes no significant increase in nanoparticles diameter. This can be explained by special hindrance exposed by HSA molecules.
DTT content
No significant changes appeared in nanoparticles size by increasing the amount of DTT. It can be concluded that small amounts of DTT can activate enough HSA molecules for reacting with thiol groups in PLGA-PMBH polymers.
Organic to aqueous phase volume ratio
The ratio between internal and external phases showed a small effect on size of NPs. By increasing the organic to aqueous phase percent from 5 to 20 %, a slight decrease appeared in size of NPs. Low polymer viscosity produces smaller organic droplets. The coalescence of organic droplets can be prevented by a large amount of organic solvent available for diffusion [20].
Differential scanning calorimetry (DSC) The DSC results in ● ▶ Fig. 2 provide quantitative information
about the physical state of the drug in NPs. Pure PLGA exhibited an endothermic peak at 60 °C which refers to the glass transition temperature of polymer. No melting point was detected, because
Darvishi B et al. Glycyrrhetinic Acid Loaded Nanoparticles … Drug Res
Fig. 2 DSC thermograms of PLGA, HSA, GLA and GLA loaded PLGA-HSA NPs.
PLGA appears amorphous in nature [21]. Pure HSA shows an endothermic peak at 70 °C which is due to its melting point [22]. Pure GLA had an endothermic peak at 300 °C corresponding to its melting point. The DSC curve of GLA, HSA and PLGA in their physical mixture shows the peaks resulting from overlapping of the DSC curves of the separated components. Thus, no interaction and crystalline change could be attributed to the heating process. The DSC curve of GLA-loaded NPs shows the event corresponding to the glass transition temperature of PLGA (60 °C) and also the peak in correspondence to the melting point of albumin (70 °C) which confirms the existence of albumin in the structure of NPs. In GLA-loaded NPs, the endothermic peak of GLA was absent. DSC studies did not detect any free GLA in the GLA-loaded NPs sample. Thus, it can be concluded that the drug incorporated into NPs was in an amorphous state in the polymer
DrugRes/2014-04-0683/14.1.2015/MPS
Original Article
In-vitro release studies
Fig. 4 GLA release pattern from S1 and S2 in PBS medium (pH = 7.4).
matrix. This confirmed the molecular dispersion of GLA in the polymer matrix.
Fourier-transform infrared spectroscopy
FT-IR spectra of PLGA, HSA and HSA conjugated PLGA NPs are shown in ● ▶ Fig. 3. HSA spectrum shows characteristic peaks at 3 430, 3 062, 1 652, and 1 531 cm − 1 which are assigned to the stretching vibration of –OH, amide A (mainly –NH stretching vibration), amide (mainly –C = O stretching vibrations), and amide (the coupling of bending vibrate of N–H and stretching vibrate of C–N) bands, respectively. Pure PLGA sample shows peaks such as –CH, –CH2, –CH3 stretching (2 850–3 000 cm − 1), carbonyl –C = O stretching (1 747 cm − 1), C–O stretching (1 050–1 250 cm − 1), and –OH stretching (3 200–3 500 cm − 1). When HSA conjugated PLGA NPs spectrum was evaluated, 1 750 cm − 1 (carbonyl –C = O stretching of ester) and 1 643 cm − 1 (carbonyl –C = O stretching of amide) absorption peaks were observed which confirms the presence of both PLGA and HSA components in the conjugated NPs [23].
The in-vitro release profile of the pure GLA and GLA-loaded NPs with different PLGA contents are shown in ● ▶ Fig. 4. The time needed for dissolving 50 % of drug (t50 %) which is inversely related to the release rate, are: 3 h for pure GLA, 19.5 h for GLAloaded NPs prepared by 5 mg PLGA, and 67 h for GLA-loaded NPs prepared by 20 mg PLGA. It is evident that an increase in the amount of polymer in the nanoparticles would cause a more reduction in release rate of GLA from NPs. ▶ Fig. 4 shows that the release of GLA from NPs was slower and ● more sustained than that of pure GLA. The rate of drug release from any solid or semi-solid delivery system is usually controlled by dissolution and/or diffusion [24]. Here in, the release profiles of GLA from nanoparticle formulations could be divided in 2 phases: an initial burst release for 4 h followed by a very slow release pattern for the rest of release profile. The quick first release of GLA is ascribed to the dissolution and diffusion of the drug that was poorly entrapped in the polymer matrix, while in the longer time periods the GLA molecules that are more tightly entrapped to the polymer matrix are released. The amount of initial burst release depends upon the amount of PLGA in the nanoparticle samples. The results showed that during the first 4 h, an initial burst release led to an early release of 49.8 and 34.2 % of drug from NPs with 5 and 10 mg PLGA, respectively.
In vitro cytotoxicity studies
HepG2 cell line was used in order to assess the in vitro cellular cytotoxicity of the samples. HepG2 cell line is considered as one of the suitable models for cytotoxicity studies [25]. Following 72 h of exposure to the GLA-loaded NPs and free GLA, cell viabil▶ Fig. 5). The cytotoxicity results ity was assessed by MTT assay ( ● are presented in ● ▶ Table 2. The IC50 of free GLA, and GLA-loaded NPs were 83.4 ± 0.56 and 9.2 ± 0.21 µg/mL respectively which is approximately 9 times more toxic after 72 h period of incubation in the cell line. The result indicates that the GLA formulated in the PLGA-HSA NPs has shown advantages in achieving lower cell viability, and or equivalently higher cytotoxicity, vs. free GLA. When GLA is
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Fig. 3 FT-IR spectrum of HSA, PLGA, their physical mixture (HSA and PLGA) and PLGA NPs conjugated with HSA.
DrugRes/2014-04-0683/14.1.2015/MPS
Original Article
has been revealed that NPs are able to be endocytosed by phagocytic cells and release the drug molecules inside the cells [19]. GLA-loaded NPs could be useful in drug targeting to the phagocytic cells to improve the treatment of intracellular infections compared to the treatments using free antibiotics. Other NPs have been evaluated for ocular [28] and oral [29] administration, which suggests that the formulated GLA-loaded NPs could be useful for the mentioned routes of administration.
Conclusion
Table 2 IC50 of GLA, GLA loaded NPs and drug free NPs against Hep-G2 cell line. Sample
IC50 (µg/l)
GLA GLA-loaded PLGA-HSA NPs Drug free NPs
83.4 ± 0.56 9.2 ± 0.21 –
Table 3 Minimum inhibitory concentrations (μg/l) of GLA, GLA loaded NPs and drug free NPs against selected bacteria. Sample GLA GLA-loaded PLGA-HSA NPs Drug free NPs
S. aureus 150 75 –
S. epidermidis 37 9.3 –
P. aeruginosa 75 18.7 –
In conclusion, an albumin-conjugated PLGA NPs of GLA was developed that had higher anticancer and antibacterial activity than free drug. HSA conjugation to PLGA NPs was achieved through disulfide bond formation between the para-maleimido benzoic hydrazide derivative of PLGA and HSA. Different nanoparticles with various drug to polymer ratio, HSA content, DTT content, and acetone volume were evaluated. The drug to polymer ratio had significant effect on the size and morphology, the drug loading, and release profile. It was shown that GLA-loaded PLGA-HSA NPs, provided desirable drug loading and size characteristics making them suitable for i. v. administration. The developed nanoparticles presented more than 9 times cellular mortality compared to free GLA. The release studies indicated that after the initial burst release, controlled release of GLA continued for more than 6 days; this release profile could be the ideal for antibacterial application.
Conflict of Interest
▼
The authors declare no conflict of interest. encapsulated in PLGA-HSA NPs, a higher GLA concentration is transported to the intracellular space which may be due to the small size of NPs and the passive targeting of macromolecules resulted from EPR effect. As a result a better efficacy and a lower IC50 is observed for PLGA NPs conjugated with HSA [21]. The outer layer of PLGA-HSA NPs, forms a stealth surface with a threshold gap that can reduce the NPs interaction (both between NPs and between cells and NPs) [22, 26]. Similar MTT assay was performed with PLGA-HSA NPs without encapsulating GLA to assess any intrinsic toxicity. GLA-unloaded NPs did not show any cytotoxic effect (data not shown).
Antimicrobial activity of the nanoparticle suspensions
The MIC of GLA-loaded NPs and free GLA on S. aureus, S. epider▶ Table 3). GLAmidis, and Ps. aeroginosa are presented in ( ● unloaded NPs showed no antibacterial activity which indicates that none of the ingredients of formulation have any antibacterial effect. The MIC of GLA-loaded NPs were approximately 2 times lower on S. aureus, 3 times lower on S. epidermidis, and 4 times lower on Ps. aeruginosa compared to free GLA. This indicates that the effective dose of this antibiotic can be reduced against the above bacteria and hence the side effects of the drug will reduce. Consequently, in vitro anti-microbial activity of GLA-loaded NPs was better than the free drug. In other studies enhanced antimicrobial activity from PLGA NPs containing antimicrobial agents has been reported [19, 27]. This may be related to the improved penetration of NPs from biological membranes. For GLA to be effective it should pass through the bacterial membrane in order to inhibit the synthesis of DNA and RNA. It Darvishi B et al. Glycyrrhetinic Acid Loaded Nanoparticles … Drug Res
Affiliations 1 Department of Pharmaceutics, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran 2 Nanotechnology Research Center, Tehran University of Medical Sciences, Tehran, Iran 3 Department of Chemistry, Amirkabir University of Technology, Tehran, Iran 4 Drug and Food Control Department, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran 5 Department of Medicinal Chemistry, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran
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▼ Fig. 5 Cell viability in HepG2 cell line in exposure to free GLA and GLA encapsulated PLGA-HSA with various concentrations against Hep-G2 assessed by MTT assay (n = 3).
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