INTRODUCTION:

The efficacies of many promising drugs are often limited by their potential to reach their therapeutic site of action when delivered by the conventional drug delivery systems. In contrast, a targeted drug delivery would not only increase the amount of drug reaching the site but also simultaneously decrease the amount being distributed to other parts of the body¹ Thus, it supplies the drug selectively to its site of action in a manner that provides maximum therapeutic activity (through controlled and predetermined drug release kinetics), prevents degradation or inactivation during transit to the target sites and protects the body from adverse reactions. Nanoparticles are solid colloidal particles ranging in size from 10 nm to 1000 nm (1 micron), which hold great promise as targeted drug delivery systems, especially for intracellular infections. They consist of macromolecular materials in which the drug is dissolved, entrapped, or encapsulated, and/or to which the drug is adsorbed or attached²

As nanoparticles are passively taken up by the phagocytic cells and find access to the lysosomes, the research aimed at developing nanoparticulate targeted delivery systems of selected anti-infective drugs for passive targeting to the macrophages of the human body, resulting in improved therapeutic efficacy of these drug(s) for the treatment of several difficult to treat and dreaded intracellular diseases³

Traditionally, fluoroquinolones especially ciprofloxacin has been employed in the treatment of many intracellular bacterial infections. However, research has proved that ciprofloxacin is less effective than other higher generation fluoroquinolones due to its low intracellular penetration and relatively short intracellular residence time leading to subsequent emergence of resistance⁴.The higher generation fluoroquinolones though promising, have been associated with serious toxic effects. Thus, ciprofloxacin was considered as an ideal testing (quinolone) candidate for incorporation into colloidal carriers such as nanoparticles to overcome the difficulties of intracellular bacterial therapy⁵

Formulation of Nanoparticles – An Overview

Nanoparticles from preformed polymers can be prepared by several methods of preparation which are classified into emulsification – based methods and direct precipitation – based methods. Depending upon the characteristics of the drug to be incorporated and the polymer(s) different methods are employed for the preparation of nanoparticles. The salting out method, though feasible is generally not employed for the formulation of nanoparticles in a laboratory scale, because it requires intense purification step (removal of salting agent) by cross flow filtration technique.

Emulsification diffusion method produces satisfactory nanoparticles but again is not employed for the preparation of nanoparticles, because nanoparticles prepared by this method cannot be freeze-dried due to the presence of benzyl alcohol in the organic phase. Nanoparticles as dispersions have a drastically low shelf life when compared to the freeze-dried forms and hence is not viable commercially. Thus, out of the five feasible methods of nanoparticle preparation, only three methods were found to be suitable, viz., Emulsification – based methods as 1.Solvent Emulsification Evaporation method, 2.Spontaneous Emulsification Solvent Diffusion method. Direct precipitation – based method as 3. Solvent Displacement method.

MATERIALS AND METHODS:

Materials

Poly D,L-lactide-co-glycolide (PLGA) and Poly D,L-lactide (PLA), Poloxamer 188 are gifted by Fourrts (India)Lab. Pvt Ltd, Chennai. Ciprofloxacin gifted by KAPL , Bangalore. D-(+)-Glucose monohydrate purchase from SD fine chemicals, dichloromethane and acetone purchased HPLC grade from Qualigens. Double distilled demineralized water (Millipore, India). And other chemicals were used are analytical grade.

Preparation of Ciprofloxacin PLGA/ PLA Nanoparticles

The nanoparticles were prepared with 100 mg of the polymers PLGA and PLA along with 50 mg of ciprofloxacin were dissolved in 20 ml of methanol: acetone in the ratio 1: 3, which formed the organic phase. This organic phase was poured through an orifice of size (0.45 mm) at the rate of 1 ml/min under atmospheric pressure into an aqueous medium (40 ml) containing poloxamer 188 as the stabilizer under moderate magnetic stirring (1000 rpm). After the addition of the organic phase, stirring was continued for 30 minutes at the same speed. After 30 minutes, the colloidal dispersion was subjected to heating under reduced pressure to remove acetone and the solution concentrated to 20 ml. Thus obtained dispersion was ultra centrifuged and the supernatant was analyzed for the amount of free drug. The pellet obtained after ultracentrifugation was freeze-dried in a lyophilizer (Yorco, India) after resuspending in a lyoprotective solution of 20% glucose (20 ml). Freeze-dried nanoparticles were then subjected to drug incorporation studies, surface morphology using SEM (Jeol, Japan).

Optimization of Ciprofloxacin PLGA and PLA Nanoparticles

Variable 1: Polymer (PLGA/PLA) – 25 mg (-1) and 75 mg (+1), Variable 2: Stabilizer – Poloxamer 188 – 0.5% (-1) and 1.5% (+1). Response: Drug incorporation – Target 50 mg (100%). Design: Response Surface methodology – CCD (Central composite design).

Surface Morphology

The particle morphology of the nanoparticles was observed by Scanning Electron Microscopy – SEM (Jeol, Japan)⁶ The freeze-dried nanoparticles were placed on a graphite surface and coated with gold using an ion sputter (Jeol, Japan) and observed in the electron microscope at 12 kV.

Particle Size and Size Distribution

The particle size distribution studies of the nanoparticle formulations were carried out by laser diffraction method employing Malvern MasterSizer, UK⁷. The freeze-dried nanoparticles were reconstituted and diluted (till the obscuration was between 10% - 30% or 0.1 – 0.3) with double distilled demineralized water (Millipore, India). The reconstituted and diluted dispersions were added into the sample holder of the mastersizer and the particle size distribution was recorded.

Table 1: Experimental design and drug incorporation values of ciprofloxacin PLGA nanoparticles

Sl. No.	Run	Variable 1 - PLGA (mg)	Variable 2 - Poloxamer 188 (%)	Response - Drug incorporation (mg)
1	9	25.00	0.50	15.0
2	13	75.00	0.50	26.0
3	4	25.00	1.50	18.5
4	6	75.00	1.50	28.5
5	5	14.64	1.00	13.36
6	1	85.36	0.29	34.90
7	14	50.00	1.71	20.95
8	12	50.00	1.00	20.95
9	10	50.00	1.00	18.95
10	10	50.00	1.00	19.95
11	5	50.00	1.00	20.00
12	5	50.00	1.00	19.85
13	3	50.00	1.00	19.95

Table 2: Experimental design and drug incorporation values of ciprofloxacin PLA nanoparticles

Sl. No.	Run	Variable 1 - PLA (mg)	Variable 2 - Poloxamer 188 (%)	Response - Drug incorporation (mg)
1	9	25.00	0.50	12.40
2	8	75.00	0.50	23.30
3	4	25.00	1.50	17.60
4	2	75.00	1.50	24.90
5	12	14.64	1.00	10.50
6	3	85.36	0.29	31.00
7	5	50.00	1.71	18.81
8	6	50.00	1.00	19.60
9	7	50.00	1.00	18.15
10	10	50.00	1.00	18.20
11	1	50.00	1.00	18.20
12	11	50.00	1.00	18.22
13	13	50.00	1.00	18.23

Drug Loading Efficiency

To determine the drug contents, Freeze-dried nanoparticles were dissolved in 10 ml of benzyl alcohol (solvent in which both the drug and the polymer dissolves) and filtered through 0.2 μm filters (Millipore, India). The amount of drug in the filtrate was spectrophotometrically estimated at 322.5 nm and 324.5 nm for ciprofloxacin. Prior studies had established no interference from the polymer under the given conditions of wavelength.

Drug Content (% w/w) = [weight of drug in nanoparticles /weight of nanoparticles recovered] x 100

Drug Incorporation (%) = [amount of drug in nanoparticles / amount of drug used in formulation] x 100

Separation of Free (Un-Entrapped) Drug

The nanoparticle dispersions were first filtered through 1 µm filters (Whatman, Japan) and then subjected to ultracentrifugation 3 times at 8000 g for a period of 30 minutes each time in a Remi RM-12C micro centrifuge.The supernatant containing the free drug was separated from the pellet and filtered through 0.22 µm filters (Millipore, India). The amount of free drug in the supernatant was spectrophotometrically estimated at 277.5 nm for ciprofloxacin. The pellet obtained after ultracentrifugation was redispersed with a lyoprotective solution of 20% glucose (20 ml) and freeze-dried (Yorco lyophilizer, India) for 16 hours

RESULTS AND DISCUSSION:

Preparation of Ciprofloxacin PLGA and PLA Nanoparticles

Nanoparticles of ciprofloxacin employing PLGA and PLA polymers were prepared by the solvent displacement method (direct precipitation – based method). The other emulsification – based methods could not be employed for the preparation of ciprofloxacin nanoparticles because ciprofloxacin was not soluble in dichloromethane and acetone which are the solvents of the organic phase.

Optimization of Ciprofloxacin PLGA and PLA Nanoparticles

Thirteen nanoparticle formulations were prepared as per table 1 and2 to elucidate the effect of the two independent variables; concentration of polymer (PLGA/PLA) and concentration of the stabilizer (poloxamer 188). The dependent response variable measured was the drug incorporation. Experiments were conducted in random sequence and the center points were repeated five times in order to evaluate the experimental error.

The drug incorporation efficiency was considerably low and therefore, it was felt necessary to carry out mathematical optimization studies to improve the drug incorporation efficiency in both the cases. The response to be optimized was fixed as ‘drug incorporation’ since it is the most important parameter along with particle size for the success of the nanoparticle formulation(s) as targeted drug delivery system. As the particle size of all the formulations were around 500 nm, and within the limits (< 1000 nm), optimization with particle size as the response was not required.

Optimization of the method of preparation was performed by response surface methodology (RSM) (specifically; randomized rotatable central composite designs – CCD) as it is the most suitable technique for the modeling and analysis of problems in which the response of interest is non-linear and influenced by several variables⁸. The mathematical modeling was carried out by employing the software – Design Expert V 6.0.5 of the Statease Inc., USA.

Table 3: ANOVA for ciprofloxacin PLGA nanoparticles (Quadratic model)

Sum of squares

Mean square

F value

Prob > F

Model

PLGA

Poloxamer 188

(PLGA)²

(Poloxamer 188)²

PLGA x Poloxamer188

415.00

370.82

12.24

32.35

1.63

2.09

83.46

369.88

12.00

33.25

1.60

2.05

42.35

187.32

6.20

16.35

0.81

1.04

<0.0001

0.0410

0.0045

0.3925

0.3368

R² – 0.9679

Table 4: ANOVA for ciprofloxacin PLA nanoparticles (Quadratic model)

Sum of squares

Mean square

F value

Prob > F

Model

PLA

Poloxamer 188

(PLA)²

(Poloxamer 188)²

PLA x Poloxamer188

298.11

279.20

7.85

9.28

0.93

3.15

60.15

278.36

7.90

9.28

0.89

3.01

21.80

100.85

2.85

3.41

0.32

1.18

0.0004

<0.0001

0.1329

0.1079

0.5828

0.3152

R² – 0.9405

Ciprofloxacin PLGA Nanoparticles

The drug incorporation values along with the run order are shown in table 1.The results were fitted to quadratic model of regression as it showed the maximum values of R² and model sum of squares. ANOVA was performed and the results are shown in table 3. ANOVA proved that the model was significant (with a probability F value of <0.0001). PLGA concentration most significantly affected the drug incorporation as indicated by a probability F value of <0.0001.

The polynomial equation giving the mathematical relationship between each of the factors was found to be: Drug incorporation = 11.383 - 0.014 (PLGA) + 1.492 (poloxamer 188) + 0.0034 (PLGA)² + 1.936 (poloxamer 188)² – 0.057 (PLGA x poloxamer 188).

The three-dimensional response surface graph along with the contour graph was plotted (figure 1), followed by numerical optimization and model validation. The 3D

Table 5: Yield / Nanoparticle Recovery

Sr.

No.

Nanoparticles formulations

Wt of polymeric material + drug + stabilizer taken (g)

Wt of nanoparticles obtained* (g)

Nanoparticles recovery (%)

SDP-C-OPTA

SDL-C-OPTB

0.723

0.785

0.712 ± 0.003

0.725 ± 0.003

98.47

92.35

Table 6: Amount of free drug in nanoparticles formulations

Sr. No.

Formulation code

Amount of free drug* (mg ± SD)

Amount of free drug (%)

SDP-C-OPTA

SDL-C-OPTB

6.20 ± 0.05

6.59 ± 0.05

12.38

13.20

Table 7: Drug content and drug incorporation of nanoparticles formulations

Sr. No.

Nanoparticles

Formulations

Wt of nanoparticles obtained* (mg)

Wt of drug in nanoparticles** (mg)

Drug content (% w/w)

Drug incorporation (%)

SDP-C-OPTA

SDL-C-OPTB

712

725

43.03 ± 0.15

43.12 ± 0.30

6.03

5.94

86.05

86.23

Table 8: Optimized formulae and formulation codes of ciprofloxacin PLGA and PLA nanoparticles

Formulations	Drug	Polymer and its concentration	Poloxamer 188 concentration	Formulation code
Ciprofloxacin nanoparticles (solvent displacement method)	50 mg	PLGA – 113 mg	560 mg (1.4%)	SDP-C-OPTA
		PLA – 135 mg	600 mg (1.5%)	SDL-C-OPTB
		Organic Phase: Acetone – 15 ml + Methanol – 5 ml Aqueous Phase: Water – 40 ml

Table 9 : Particle size distribution parameters of nanoparticles formulations

Sr. No.

Nanoparticles Formulations

Volume mean diameter (D[4,3]) μm

Volume median diameter

(D[v 0.5]) μm

Volume median diameter

(D[v 0.9]) μm

Mode

SDP-C-OPTA

SDL-C-OPTB

0.525

0.540

0.44

0.46

1.01

1.08

0.46

0.49

Figure 1: 3D – Response surface graph of ciprofloxacin PLGA nanoparticles

response surface graph substantiated the fact that PLGA concentration most significantly affects the drug incorporation. From the numerical optimization results (table no 8), the optimized formula for the preparation of ciprofloxacin PLGA nanoparticles were selected by maximum drug incorporation.

Figure 2: 3D – Response surface graph of ciprofloxacin PLA nanoparticles

Ciprofloxacin PLA Nanoparticles

The drug incorporation values along with the run order are shown in table 2. Even here the results were fitted to quadratic model of regression as it showed maximum values of R² and model sum of squares. ANOVA was performed and the results are shown in table 4. ANOVA proved that the model was significant, with a probability F value of 0.0004. PLA concentration most significantly affected the drug incorporation as indicated by a probability F value of <0.0001. The other variable poloxamer 188 did not significantly affect the response as evidenced by a probability F value > 0.05 (0.1332).

The polynomial equation giving the mathematical relationship between each of the factors was found to be: Drug incorporation = 6.939 + 0.122 (PLA) + 2.680 (poloxamer 188) + 0.0018 (PLA)² + 1.444 (poloxamer 188)² – 0.071 (PLA x poloxamer 188).

Figure 3 SEM photomicrograph of ciprofloxacin PLGA nanoparticles

Figure 4 SEM photomicrograph of ciprofloxacin PLA nanoparticles

Even the three-dimensional response surface graph along with the contour graph showed that only variable PLA significantly affects the response and not poloxamer 188 (figure 2). From the numerical optimization results (table no 8) is an obtained formula for the preparation of ciprofloxacin PLA nanoparticles due to its maximum drug incorporation.

Characterization of Ciprofloxacin PLGA / PLA Nanospheres

The nanoparticle recovery results are shown in table 5. Varied between 90 to 98% for the formulations prepared. The drug, which is added during the preparation can either get entrapped or remain (un-entrapped) free. This free drug has to be removed for all further studies to avoid misinterpretation of the data. The observations are given in the following table 6, which is range about 12-13%(w/w).The drug content and the drug incorporation were calculated as per the formulae given in table 7 of having around 86%(w/w) .The surface morphological studies revealed that the nanoparticles obtained had a smooth surface and were spherical in all the formulations. Also the particles were discreet (non-aggregated).The average particle size / volume mean diameter (D [4, 3]) and volume median diameters – (D[v, 0.5]) and (D[v, 0.9]) of the nanoparticle formulations are given in the table 9. D[4,3] is the volume mean diameter, it is the diameter of the sphere having the same volume as our real particle. This volume mean diameter denotes the average volume size of the spheres under consideration. Laser diffraction method of particle size analysis always calculates a distribution based around the volume term and hence this value is taken as the average particle sized [v 0.5] is the volume median diameter, it is this value of the particle size which divides the population exactly into two equal halves i.e., there is 50% of the distribution above this and 50% below.

CONCLUSION:

The current study states that ciprofloxacin encapsulated PLGA and PLA Nanoparticles were prepared by solvent displacement technique by using the numerical optimization techniques was found to effectively investigated to choose polymer concentration and drug content ration PLGA / PLA nanoparticles which shown spherical nature. Particles size range from 525-540 nm. Loading efficiency was found approximately 86%w/w. Nanoparticles can be better suitable method to prepare by optimization method than trial error methods to minimize the prediction of polymer and drug experimental conditions.

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Received on 31.01.2009 Modified on 28.02.2009

Research J. Pharm. and Tech. 2(1): Jan.-Mar. 2009; Page 186-190