Formulation optimization of pH-sensitive liposomes based drug delivery of Carboplatin and anti-proliferative evaluation against A549 (human lung carcinoma) cell lines

Abstract

The developments of pH–sensitive liposomes which are stable at physiological pH i.e. (6.8–7.4) have not explored much up until now. These lipid vesicles will go through destabilization and attain fusogenic properties in acidic conditions leading to liberation of aqueous contents. Carboplatin, included in the family of alkylating agent was found to exhibit adverse effects like myelo suppression, ion thrombocytopenia and leucopenia. Therefore, in order to circumvent these effects, carboplatin pH-sensitive liposomes for specific delivery is the ideal criteria and it poses a great challenge since the water-soluble drugs exhibited very low entrapment efficiency. The essential portion of study was evaluated using the Design Expert software 8. The pH-sensitive liposomes were optimized using Central composite design and one factor Response surface model design method and were prepared by film hydration method. Two formulation variables like drug: lipid ratio (X₁) and volume of hydration media (X₂) used to vary at three different levels and the other three variables viz. temperature, speed of rotation and vacuum applied were kept constant. The Response surface and contour plots were figured to elicit the effects of interaction of variables on the overall entrapment efficiency. pH-sensitive liposomes of carboplatin have been regarded as a promising delivery systematic approach in order to target tumor tissue as evaluated by the pre-clinical studies in both in vitro and ex-vivo conditions.

Keywords

Liposomes formulation factorial design drug delivery system anticancer

Abbreviations

Phosphatidylcholine

DOPE

Dioleoylphosphatidylethanolamine

Phosphatidylethanolamine

EPC

EggPhosphatidylcholine

CHEMS

Cholesterylhemisuccinate

MTT

3-(4, 5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide

CDH

Central Drug House

PBS

Phosphate buffer saline

Phosphate buffer

FTIR

Fourier transforms infra-red

CCD

Central composite design

RSM

Response surface model

AUC

Area under the curve

ELISA

Enzyme-linked immunosorbent assay

NCCS

National center for cell science

Standard deviation

SEM

Scanning electron microscopy

CDR

Cumulative drug release

Cpt

carboplatin

Cpt-DPF

carboplatin dry powder formulation

TFHM

Thin film hydration method

ICH

International conference on harmonization

1 Introduction

Liposomes became justified to save the facet effects of the integrated drug in addition to enhance the efficacy of the drugs. The detrimental results linked with anticancer formulations make them suitable applicants for the site specific delivery. The blood vessel in tumor tissue is permeable because its basement membrane is sporadic having gaps within the range from 100–780 nm.The liposomes can extravagate and gather in the interstitial space as a result of improved permeation and retention effect (EPR impact). The passive targeting accomplishes carriers having diameter greater than 100 nm which are perfect for EPR effect. Maximum chemotherapeutic agents have more extent of distribution on intravenous management which frequently ends in a narrow therapeutic index due to excessive degree of toxicity in healthy tissues [1]. For that reason, entrapment of drugs in macromolecular carriers has been done by inclusion of liposome, where the volume of distribution gets attenuated and leads to reduced nonspecific toxicities and an enhancement in the amount of drug that may be efficiently introduced to the tumor [2 –4]. The development of pH–sensitive liposomes which are solid at physiological pH i.e. 6.8–7.4 had not been much exposed, till now. These lipid vesicles will go through destabilization and accumulate fusogenic under acidic conditions leading to the release of aqueous contents from inside. In other words, fusogenic liposomes fuse with biological membranes for enhancing drug contact and delivery of liposomes into cells. The tumor vasculature in a growing tumor is leaky, pH-sensitive liposomes will generally tend to get trapped inside the tumor due to EPR effect and consequently, it could enhance antitumor drug efficacy.

Since carboplatin belong to this class and found to be less nephrotoxic than cisplatin. Carboplatin in liposomes is justified to prevent its adverse effects and has been used to increase the therapeutic efficiency like myelosuppression, thrombocytopenia and leucopenia [5 –7]. Furthermore, water soluble carboplatin loading helps in improving the percent drug entrapment and has been taken as variable parameters in this study.

2 Experimental section

2.1 Optimization using factorial design

The word “optimization” in arithmetic and computer technology or mathematical programming, refers to choosing the high-quality detail from a few sets of available alternatives. Inside the most effective case, this indicates solving troubles wherein one seeks to reduce or maximize a real characteristic by using systematically selecting the values of real or integer variables from inside an allowed set. The critical portion of evaluation turned into the usage of the layout expert software program 8. Furthermore, it detects the interactions among the variables influencing the quantity of drug loading in liposomes with different reaction parameters. At last, in keeping with the final effects, this program suggested some formulations and additionally expected their responses containing a possibility component named “Desirability” that ranged between 0-1 in the subsequent step, importance of this affect became additionally statistically confirmed via factorial ANOVA check (P < 0.05) [8]. There are various factorial designs likewise, Factorial layout, Fractional factorial layout, central composite design, Plackett Burman layout, star design, box behnken design, Equiradial design, Taguchi layout, and many others. The variety of impartial variables selected estimates the variety of experiments which might be performed inside the laboratory [9].

Out of all designs, central composite design and one factor response surface model design (RSM) has been selected for optimization.

2.1.1 Central composite design

An important composite design is an experimental layout, beneficial in response surface method, for building a 2d order (quadratic) model for the reaction variable while not having use of whole three-degree factorial experiment. CCDs are very efficient, supplying a whole lot records on test variable consequences. CCDs are very flexible. The availability of several forms of CCDs allows their use below extraordinary experimental areas of operability.

2.1.2 One factor RSM design

It lets us to develop a cubic model for one numeric issue. Five levels of a single factor (–1, –0.5, 0, 0.5, 1) and replicates allow a lack of fit and pure error determination for a quadratic model [10]. The conventional strategies of optimization do not offer efficient effects. In the optimization, use of 3³ factorial designs is difficult to assess i.e. tedious system. The pH- sensitive liposomes were optimized by the use of central composite design and one factor response surface model design. The two degree complete factorial or fractional factorial designs augmented through number of middle points and different selected runs had been considered. The used layout allowed the estimation of all the parameters required to fit a linear order or Quadratic version to a given reaction. One element RSM design allowed became a cubic version for one numeric factor. Five stages of a single variable (–1, –0.5, 0, 0.5, 1) and replicates permit a lack of fit is added to the overall amount of unexplained variance. The prevailing work accomplishes the formation of pH-sensitive liposomes by film hydration technique which becomes optimized by the use of valuable composite layout and further via one response surface model layout. Method variables like drug: lipid ratio(X1) and quantity of hydration media(X2) used to vary at three different degrees and the opposite three technique variables including temperature, speed of rotation and vacuum applied were kept constant. The response surface and contour plots have been figured to elicit the effects of interaction of variables on the entrapment efficiency [10].

2.2 Materials and methods

2.2.1 Chemical substances

Carboplatin was gift sample from Fresenius Kabi Oncology Limited, India. Egg Phosphatidylcholine(EPC) and Cholesterylhemisuccinate(CHEMS) was bought from Sigma Aldrich, Dioleoylphosphatidyl ethanolamine become acquired as gift sample from Lipoid, GmbH, Germany. Chloroform turned into purchased from Merck, India. Dextrose 5% w/v (D5) became acquired from market preparation. MTT, HAMF-12 Media, L-glutamine Penicillin, Streptomycin, Dimethyl sulfoxide, Terhalose and Triton X were received from HiMedia Laboratories (Mumbai). The reagents used are of analytical standard.

The preformulation research had been accomplished in order to pick the proper form of drug substance (carboplatin), evaluation of its physical properties and thorough knowledge of materials stability which elicited the development of the most efficient drug delivery system.

2.2.2 Preparation of Calibration curve of carboplatin in PBS (7.4) and in PB (5.5)

Appropriate aliquots of stock solution of carboplatin (1 mg/ml) were taken in volumetric flasks and had been diluted up to a specific volume with PBS (7.4) and PB (5.5). A specific concentration of carboplatin solution changed into organized in the range of 10–50 mg/ml and further evaluated their absorption maximum (λ_max). The λ_max has been determined for 10μg/ml solution towards blank on UV-visible Spectrophotometer between the wavelength range of 190–400 nm. The readings of absorbance±SD were recorded in triplicate as shown in Fig. 1. Second order spectra of carboplatin become additionally determined with the usage of PBS (7.4) for greater accurate effects and the observed λ_max changed in comparison with the standards [11] as shown in Fig. 2a and 2b.

Fig. 1

(a) Absorption spectrum of carboplatin in PBS (pH-7.4). (b) Absorption spectrum of carboplatin in PBS (pH-5.5).

Fig. 2

(a) Curve depicted as second order spectra of carboplatin. (b) Experimentally obtained second order spectra of carboplatin. (c) FTIR spectra of carboplatin.

2.2.3 FTIR Spectra of carboplatin

FTIR Spectra had been analyzed between a wave range locations of 400–4000 cm^–1 using Nicolet omnic software program. The characteristic peaks were determined and compared with the old peaks for the identity of the drug as shown in Fig. 2c.

2.2.4 Melting point of carboplatin

The melting point is determined in a capillary tube. The expression “melts approximately. . . ” manner that the temperature at which the substance is absolutely melted, as indicated with the aid of the disappearance of the drug, may be within the variety±4°C from the stated, until in any other case indicated. Melting point can be determined by the usage of capillary in melting point apparatus [12]. The carboplatin (5 mg) was taken in capillary tube, closed at one end. The melting-point apparatus was heated to a temperature 5–10°C below the predicted temperature of melting and the heating changed into adjusted so that the temperature in the chamber raised about 1°C consistently. The capillary has been introduced with the substance into the heated chamber, and the temperature was mentioned whilst the sintered substance has become absolutely transparent.

2.2.5 Apparent partition coefficient of carboplatin

Partition coefficient is the ratio of concentrations of compound inside the levels of a combination of immiscible solvents at equilibrium. Apparent partition coefficient was determined which elicited approximate results. It was calculated using shake-flask technique. The weighed amount of carboplatin (10 mg)dissolved in water and n-octanol aggregate (1:1) [13]. The aggregate became stored for 72 hours with occasional stirring. Water, n-octanol became separated using separating funnel and analyzed for drug content with the help of UV Spectrophotometer at predetermined value λ_max suitable for carboplatin.

The obvious partition coefficient changed into calculated with assist of formula given underneath:

Apparent Partition coefficient = (concentration in n-octanol)/ (concentration in water)

2.2.6 Solubility of carboplatin

Solubility study of the drug accomplished by dissolving extra of drug in diverse solvents and preserving them for 24 hours [14]. Then, all the solutions were vortexed on excessive pace using Spinix model 2800.Then they had been centrifuged at 5000 rpm for 10 minutes on R-8C laboratory Centrifuge and the λ_max was measured at 234 nm by the usage of Spectrophotometer [15].

2.2.7 Method of preparation

Eleven formulations have been arranged with the usage of C.C.D and seven formulations with the aid of one RSM in triplicate. The values with their coded values have been depicted in Table 1. Specific constraint values of based variables were set according of the needful method by means of the layout software program together with particle length (minimal), zeta potential = –30, PDI (minimum). Eleven batches have been prepared in line with the experimental situations as shown in Table 2. Egg Phosphatidylcholine, Dioleoylphosphatidylethanolamine (pH-sensitive lipid at specific site) and Cholesterylhemisuccinate had been weighed and dissolved in chloroform and methanol (2:1 by w/v) in a 200 ml flask. The flask was turned around by means of the rotatory evaporator at 150 rpm for 45 mins in a water bath tub at 37°C under vacuum. To the thin dried lipid film, drug solution (1.1 mg of drug) dissolved in 5% w/v dextrose solution (the usage of distinct quantity of hydration media,X2) was added and the flask became rotated again at the equal speed and temperature (40°C) but without vacuum for 90 mins for lipid film elimination and dispersion. The liposomal dispersion was poured into glass box held on ice bath for dissipation of warmth and turned into sonicated for few minutes by the use of Misonix, Ultrasonic Liquid Processors, US. The final liposomal dispersion changed into characterized for particle size, zeta analysis, and percentage entrapment performance. All the preparations have been stored inside the refrigerator and lyophilized by the use of extraordinary cryoprotectants for stability studies.

Table 1
Coded values of formulation parameters of carboplatin loaded pH-sensitive liposomes

Factors Units Levels used actual (coded)

Low (–1) Medium (0) High (1)

Drug: Lipid ratio Molar ratio 1:5 1:10 1:15

Volume of hydration media ml 2 3 4

Factors	Units	Levels used actual (coded)
Drug: Lipid ratio	Molar ratio	1:5	1:10	1:15
Volume of hydration media	ml	2	3	4

Table 2

CCD Layout

Formulation Code	Drug: lipid ratio	Volume of hydration media (ml)	Particle size±SD^* (nm)	Zeta-potential±SD^* (mV)	PDI±SD^*
1	1:5	2	382.0±4.21	–27.2±2.91	0.979±3.12
2	1:15	2	452.8±2.13	–38.0±3.11	0.398±3.23
3	1:5	4	182.0±1.19	–35.2±3.23	0.678±2.43
4	1:15	4	282.9±3.33	–30.5±4.43	0.464±3.76
5	1:5	3	384.6±3.24	–28.0±3.78	0.637±3.07
6	1:15	3	599.4±5.32	–34.4±3.11	0.125±3.45
7	1:10	2	128.6±2.91	–34.6±2.98	0.409±3.67
8	1:10	4	503.1±4.51	–37.7±3.01	0.478±4.34
9	1:10	3	267.4±2.34	–35.3±4.11	0.334±3.21
10	1:10	3	268.0±3.21	–35.6±3.45	0.321±3.01
11	1:10	3	267.8±3.12	–35.1±4.87	0.324±2.19

^*SD Standard deviation. PDI: Polydispersity index.

2.2.8 Characterization of pH-sensitive liposomes

Physical and chemical characterization of liposomes is important for the comparison of different liposome preparation. There should not be observed any major change during storage so that a well characterized product is injected and the liposomal dispersion warrants optimal reproducibility of clinical effects. The prepared liposomes containing carboplatin were characterized for following attributes. The characterization of liposomal formulation helps to determine the drug entrapment efficiency. Stability studies for the determination of percentage drug content were carried out and increase in size of the liposomes on storage over the period of one month.

2.2.9 Particle size

The mean particle size of the prepared pH-sensitive liposomes become received by way of using Zeta Sizer(Beckman coulter, Delsa Nano C) the usage of Delsa Nano 2.21. Diluted liposomal suspension changed into determined for the average particle size. The ultimate size range should be between 100–250 nm. All the measurements have been carried out in triplicate and the usual deviation were recorded [16] (Fig. 3).

Fig. 3

(a–c) Particle size analysis of pH-sensitive liposomes (n* = 3).

2.2.10 Morphology

Morphology of the pH-sensitive liposomes was ascertained from photomicrographs taken by the usage of Scanning Electron Microscope (ZEISS EVO 50) at a magnification of 100X. Image evaluation of the SEM pictures had been conducted on a set location selected on the particle flat base with a purpose to avoid tilting angle shadow impact after the powders have been covered by way of gold as shown in Fig. 4(a and b).

Fig. 4

(a) Scanning electron microscopy of carboplatin loaded pH-sensitive liposomes. (b) Photomicrograph of pH-Sensitive Liposomes.

2.2.11 Zeta potential

Zeta capacity of the liposomes became measured by the use of Zeta Sizer, Beckman coulter. The dry powder formula (dispersed in hydration media) in addition to the prepared dispersion of liposomes was analyzed by the use of Beckman coulter. All of the measurements have been carried out in triplicate and the same old deviation turned into recorded values.

2.2.12 Entrapment efficiency: Separation of loose drug from drug entrapped in Liposomes

Centrifugation technique (Retrospective method) was used for separation of free drug from liposomes. Suspension of liposomes becomes transferred in centrifuge tube. The tube changed into then centrifuged at 25000 rpm at 4°C for 40 minutes in laboratory freeze centrifuge. Free drug sediments as pellet were taken at the bottom of the centrifugation tube with the aid of this method loose drug turned into separated from the liposomal dispersion. Then free drug and the drug entrapped in the liposomal dispersion became analyzed. This method isn’t always appropriate for complete separation of the lipid part of liposomes. For that reason, potential approach was used which accomplishes the entire separation standards. This involved first passing the liposomal dispersion (10 ml) through Sephadex column G-50(already dealt with water for swelling, saved for 24 hrs and stored in 0.9% NaCl), then milky component predicted priorly become the liposomes containing drug, the transparent component turned into discovered as free drug. So, the milky dispersion (6 ml) changed into treated with Triton –X 100 and then sonicated it for 1 min by the usage of probe sonicator after which centrifuged it at high velocity of 26,000 rpm using freeze centrifuge at 4°C for 20 minutes. The λ_max of the drug entrapped in the liposomal dispersion was carried out by the usage of UV Spectrophotometer. All experiments have been achieved in triplicate [17].

2.2.13 Evaluation of entrapped drug in liposome

One ml of supernatant taken and measured the absorbance at the wavelength of 230 nm in opposition to blank on UV Spectrophotometer.

The drug entrapment can be calculated by the use of equation:

% Drug Entrapment = (Entrapped Drug)/(Drug)×100

2.2.14 In vitro drug release studies

The lyophilized powder containing ∼2 mg of carboplatin dispersed in 2 ml of buffers contained in cellophane tubing/ dialysis membrane (pretreated through washing with water). Sulfur compounds were eliminated by treating the tubing with a 0.3% (w/v) solution of Na₂S at 80°C for 60 seconds. Further, it was washed with warm water (60°C) for 2 minutes, observed by way of acidification with a 0.2% (v/v) of sulfuric acid, and finally washed with warm water for the elimination of the acid. The membrane containing the liposomal dispersion was suspended in 200 ml of buffer (Phosphate buffer, pH 5.5, 7.4) and incubated at 37°C in shaker incubator for 36 hours. The sample(1 ml) collected and changed with 1 ml of phosphate buffer(one after the other in each pH 5.5, 7.4) throughout 0.25,0.5,1,2, 3,4,5,6,8,12, 24 and 36 hours, respectively [18].

2.2.15 Solid state characterization

Lyophilization of pH-sensitive liposomes loaded with carboplatin was completed for 48 hours using only proprietary cryoprotectants, such as maltose, dextrose, trehalose, lactose, and sucrose with the usage of a lipid: sugar ratio (1:6).The flow conduct of the lyophilized powder turned into evaluated with the usage of two important parameters. The attitude of repose was measured to decide the glide properties of a powder. The compressibility index reflected the percentage voids within the powder in conjunction with the waft and dispersibility index of the system. So, the tapped density becomes evaluated by means of routinely tapping a measuring cylinder containing 10 g of powder pattern. After looking at the preliminary extent, the measuring cylinder is mechanically tapped so that no deviation in tapped volume reading has been determined. The plateau situation changed into acquired after 500 faucets for all samples. For determining the perspective of repose, the pile was cautiously constructed up by means of losing the fabric via a funnel until the end of the funnel (top, 2 cm). Piles of powder become carefully constructed up by losing the powders through a funnel tip from a peak of 2 cm [19]. Consequently, the angle of repose was calculated via inverting tangentially the ratio of peak and radius of the shaped pile. All experiments were achieved in triplicates [20]. The Carr’s compressibility index was calculated by using the measured tapped density by the use of the following equation [21]

Carr’s index = (Tapped Density-Bulk density)/ (Tapped density) *100

2.2.16 Stability studies

The optimized formulations (lyophilized samples) of pH-sensitive liposomes have been kept in desiccators at room temperature and at refrigerated situations. The samples withdrawn were rehydrated for the evaluation of the dimension’s distribution. The comparative stability studies had been achieved with capability at managed room temperature (25°C±2°C) and at refrigerated conditions (2–8°C) as per ICH recommendations. Consequently, the test has been done with three batches of identical composition for the duration of one month and the readings had been taken at exceptional time intervals i.e.0, 10, 20, 30 days by the usage of Beckman Coulter for measuring the particle size [22]. The samples withdrawn had been rehydrated for the analysis of the dimension’s distribution.

2.3 Biological activity: In vitro cytotoxicity studies

2.3.1 Cell culture and maintenance

The cell lines A549 (human lung carcinoma) obtained from NCCS Pune, India. Cell strains had been cultured in Ham’s F12 medium supplemented with L-glutamine (2 mM), Penicillin (100 units/ ml), Streptomycin (100μg/ml) and 10% (v/v) inactivated fetal bovine serum. Throughout the cultivation process, cells have been kept in a 5% CO₂ humidified incubator at 37°C.

2.3.2 Cell viability assay

It was determined by use of MTT assay [23] ELISA reader (Biorad, US, model 680). Cells have been seeded in 96 well plates (2×10⁴ cells/well) and allowed growing for 24 hours at 37°C in CO₂ incubator supplied with 5% CO₂.After 24 hours the cells were treated with the formulated pH sensitive liposomes of carboplatin at the concentration from 2–64μg/mL for 24 hours. After 24 hours, supernatants have been aspirated out and MTT dye was added to the wells and further incubated for 4 hours. After 4 hours, the violet color formazan crystals were dissolved by adding DMSO and absorbance was recorded at 570 nm by using the ELISA reader. Data was analyzed by using Microsoft excel and IC₅₀ was calculated by using Graph-pad prism.

3 Results and discussion

3.1 Optimization of pH-sensitive liposomes

The pH-sensitive liposomes were optimized by taking two factors at 3 stages, the two elements had been drug: lipid molar ratio, extent of hydration media and the reaction parameters have been particle length, zeta potential, PDI evaluated the usage of triplicate (n = 3) as defined in Table 2. $Coded variable, xi = 2 X_{i} - (X_{ih} + X_{i 1}) / X_{ih} - X_{i 1}$ (1) where X_ih and X_i1 the excessive and low value of X_i, respectively.

The polynomial equations generated by this experimental design are as follows: $Y = b_{0} + b_{1} A + b_{1} B$ (2) $Y = b_{0} + b_{1} A + b_{2} B + b_{1} AB + b_{1} 2 A + b_{2} 2 B$ (3) in which Y is the structured variable (Particle size, zeta capacity, PDI) at the same time as b₀ is the intercept, bi (b1, b2 and b3), bij(b12, b23 and b13) and bijk(b123) represents the regression coefficient for the linear and second order polynomial and A,B,C represents the levels of impartial formula variables.

The values were filled into the Equation 1 for the determination of based variables:

Particle size (length) = 338.05

Zeta potential = –35.18 - 2.03 A - 0.60 B + 3.87 A B + 3.76 A²–1.18 B²

PDI = 0.29 - 0.21 A - 0.02 B + 0.09 A B + 0.12 A² + 0.18 B²

Inside the first reaction parameter of particle size, imply model was decided on and in zeta capacity, PDI quadratic model was discovered to be decided on. The anticipated values had been calculated through the use of the mathematical version derived from the coefficients of the version evaluation as depicted in Table 3.Consequently, the outcomes of regression evaluation for particle size (Y1), Zeta potential (Y2), PD1 (Y3) i.e. Particle metrics data were given in Table 4.

Table 3

Selection of the response models

Models	Sequential p-value	Lack of Fit p-value	Adjusted R²	Predicted R²
Response (Y1)
Mean	<0.0001
Linear	0.5857	<0.0001	–0.0935	–0.7690
Second order	0.9266	<0.0001	–0.2481	–1.9674
Quadratic	0.5734	<0.0001	–0.3988	–5.8472
Response (Y2)
Linear model	0.3794	0.0037	0.0189	–1.0017
Second order	0.0165	0.0074	0.5334	0.0572
Quadratic model	0.0104	0.0274	0.8948	0.4602
Response (Y3)
Linear model	0.0388	0.0012	0.4451	–0.0677
Second order	0.3105	0.0012	0.4583	–1.2282
Quadratic model	0.0069	0.0051	0.8962	0.5329

^*p Probability value; R² Coefficient of regression.

Table 4

Particle metrics data (Particle size, Zeta potential and PolydispersityIndex for carboplatin loaded pH-sensitive liposomes)

Term	Particle size (nm)			Zeta potential (mV)			PDI
	Coefficient	SD	Range^*	Coefficient	SD	Range^*	Coefficient	SD	Range^*
Intercept	338.05	42.52	243.31–432.80	–35.18	0.60	–36.73––33.64	0.29	0.04	0.20–0.40
A-drug: lipid ratio	–	–	–	–2.03	0.48	–3.32––0.85	–0.21	0.03	–0.29–0.14
B-volume of hydration media	–	–	–	–0.60	0.48	–1.83–0.63	–0.02	0.03	–0.10–0.05
AB	–	–	–	3.87	0.59	2.37–5.38	0.09	0.04	0.00–0.19
A²	–	–	–	3.76	0.74	1.86–5.66	0.12	0.05	0.01–0.24
B²	–	–	–	–1.18	0.74	–3.09–0.71	0.18	0.05	0.07–0.31

^*SD Standard deviation. ^*The range indicates the lower and upper value of coefficients at 95% confidence interval.

Out of thirty-three formulations, the design had decided on answers and the determined criteria shown by way of greater proper system (0.69) with drug: lipid ratio (1:15), extent of hydration media (3.5 ml), expected particle size (338.05), zeta potential (–32.17), PDI (0.282). This formulation code has been decided on with the aid of the design software program due to its extra desirability factor in comparison with the alternative. The formula became demonstrated by making the preparations in laboratory three instances for concordant readings, the information stated changed into particle size (349 nm), zeta potential (–33.30) and PDI (0.272).The response surface graphs (3D surface and contour plots) have been implicated for this reason drawn (as shown in the figures) for estimating the corrected values of reaction parameters.

The second step optimization changed into the usage of one factor response surface model layout for 0.33 variable i.e.sonication time and the established variables have been particle length and entrapment efficiency. The 2 unbiased variables had been now constant by using the outcomes decided on above for drug: lipid and volume of hydration media. Five special degrees had been selected with coded values (–1, –0.5, 0, 0.5, 1) and actual values (0, 0.5, 1, 1.5, 2). Seven formulations had been organized 3 times; desirable constraints have been particle size (in minimal variety, entrapment efficiency (most) as shown in Table 5. The regressed equation of the geared-up model has been located to be linear as depicted through the design; the evaluated parameters had been linear in pattern as shown in Table 6.

Table 5

One factor RSM layout

Formulation Code	Drug: lipid ratio	Vol. of hydration media(ml)	Sonication time	Particle size	% Entrapment efficiency
S-1	1:15	3.5	0	349	74.95
S-2	1:15	3.5	1	248.3	58.5
S-3	1:15	3.5	0	235.3	66
S-4	1:15	3.5	1.5	171.1	60.1
S-5	1:15	3.5	0.5	229.3	68.4
S-6	1:15	3.5	2	151	50.4
S-7	1:15	3.5	2	154.4	45.6

Table 6

Selective criteria and suggested models by one factor RSM

Models	Sequential	Lack of Fit	Adjusted R²	Predicted R²
	p-value	p-value
Response (Y1)
Linear model	0.0156	0.9073	0.66581	0.36625
Quadratic	0.9876	0.7952	0.5823	0.04708
Cubic	0.8917	0.5539	0.4471	–0.4562
Response (Y2)
Linear model	0.0031	0.6975	0.82107	0.6826
Quadratic	0.5335	0.6129	0.79955	0.55963
Cubic	0.7302	0.4018	0.74491	0.1934

The regressed equation formed via filling the values into Equation (1) depicted as follows:

Particle size = 219.7–68.4 *A

Entrapment efficiency = 60.5–10.9*A

Coefficient values for particle size and entrapment efficiency had been decided and the nearby points has been predicted (Table 7).

Table 7

Point prediction data

Response	Prediction	SE Mean	95% CI low	95% CI high	SE Pred	95% PI low	95% PI high	99% of 95% TI low	Population 95% TI high
Particle Size	187.78	17.64	142.41	233.14	44.03	74.59	300.96	–64.87	440.43
Entrapment efficiency	55.46	1.89	50.59	60.33	4.72	43.30	67.6224	28.32	82.60

Desirability factors of formulation with validated values are:

Sonication time (1.47 min.), Particle length (187.78 nm), and Entrapment efficiency = 55.46%

The preoptimized components have been established for liposomal formulation. The calculated values for confirmed practice had been depicted in Table 8. The observed facts for particle length were detected with the aid of Zetasizer(Beckman Coulter, Delsa nano C the usage of Delsa Nano 2.21) and had been figured out as proven in Fig. 2 and entrapment performance by means of the strategies described above was measured in triplicate. The most beneficial wavelength range has been demonstrated between 100–250 nm. All the measurements had been performed in triplicate and the deviation have been recorded [24].

Table 8

Validation of the final formulation using design

Number	Sonication	Average particle	Average Entrapment
	time (in minutes)	size±SD	Efficiency±SD
		(n = 3)	(n = 3)
1	1.5	171.1	58±3.22
		180.6
		169.8
		173.8±5.89

3.2 Checkpoint evaluation

A test point evaluation turned into executed to verify the application of set up contour plots and decreased equations based on totally at the effects inferred the software program interpretation had been shown by the graphs in the Fig. 5a–5e. Linear correlation plots showed the actual response values vs. the predicted which helped to detect the values that were not easily predicted by the model. The data points were splitted evenly by the 45-degree line observed in both chosen designs.

Fig. 5

Linear correlation plot between actual and predicted responses of particle size, (5b) zeta potential,(5c) PDI using C.C.D., (5d) Linear correlation plot between actual and predicted responses of particle size (another optimized formulation), (5e) Drug entrapment efficiency.

3.3 Response surface plots

Response surface plot are helpful in providing information for the principle and the interplay consequences of two variables [25]. These plots can be easily obtained by calculating values taken by means of one constraint wherein the second varies (from –1 to 1 as an instance) with constraint of a given value. The model is related to drug: lipid ratio and volume of hydration media to the particle size, zeta potential and PDI. It’s been observed that elevated drug: lipid ratio markedly reduced the dimensions of debris, while increased quantity of hydration improved particle size drastically. Interplay among the factors did not show any arise. Likewise, with augmentation in drug: lipid ratio, Zeta capacity collapsed and with attenuation in quantity of hydration media, zeta ability elicited elevated values. In case of PDI, drug: lipid ratio showed boom responses with expanded ratios and with reduced values of volume of hydration media thereby promoted growth PDI values. So, those response surface plots had been helpful in defining the interactive effects [26]. Contour plots have been additionally drawn thereby in recommended manner plots similar in pattern with 3-D surface plots in which the later couldn’t be rotated as proven in Figs. 6a–6c and 7a-b. A contour plot allows you to visualize 3D data in a 2D plot. Contour plot constructed to establish the understanding of relationship of variables and its interaction. The effect of molar ratio and volume of hydration media on zeta potential and PDI of liposomes has been determined graphically. With augmentation in drug: lipid ratio, Zeta capacity collapsed and with attenuation in quantity of hydration media, zeta ability elicited elevated values. In case of PDI, drug: lipid ratio showed higher responses with expanded ratios and with reduced values of volume of hydration media thereby promoted higher PDI values.

Fig. 6

(a) Quadratic response model showing the influence of particle size as a function of drug: lipid ratio and volume of media used. (b) influence of zeta potential as a function of drug: lipid ratio and volume of media. (c) influence of PDI as a function of drug: lipid ratio and volume of media.

Fig. 7

(a, b) Contour plots.

3.2 In vitro cytotoxicity studies

The cytotoxicity of pH-sensitive liposomes of carboplatin was determined by using MTT assay. The results of this assay demonstrated that the IC₅₀ value of pH-sensitive liposomes of carboplatin (42μg/ml) was higher as compared to the plain carboplatin (21μg/ml) further supported the lower drug release at physiological pH and therefore more drug release at target tumor sites (in acidic pH) as represented in Fig. 8.

Fig. 8

Graph between % inhibition vs. log concentrations (IC₅₀ values of carboplatin loaded pH-sensitive liposomes and plain carboplatin).

3.3 Discussion

Carboplatin loaded pH-sensitive liposomes have been efficiently prepared through thin film hydration technique (TFHM), because it has been prepared easily within the laboratory and in contrast to different methods [27] via the usage of organic solvents and detergents.Those methodologies ought to consequently be hired effectively to any process which involves the results and interactions of many experimental variables. Furthermore, CHEMS acts as an amphiphilic stabilizer and consequently this method was anticipated to show higher stability in biological fluids. Moreover, because of the fusogenic property of a polypeptide associated with lipid bilayer membrane where such polypeptide enables fusion with, or creates a channel between, such lipid bilayer membrane and a cellular membrane, enabling transfer of a payload to or across such cellular membrane. The optimization studies were executed by the use of factorial designs i.e. CCD and one factor response model. The followed layout changed into 3² Central composite layouts priorly and one response factor design thereafter. The motive in the back of imposing the 3²(CCD) design, because it became bulky to assess 33 layouts in which 27 formulations had been made in triplicate and the dependent variables were difficult to assess in such instances. The pH-sensitive liposomes were formed when drug: lipid ratio turned into 1:15, extent of hydration media changed into 3.5 ml, sonication time was 1.5 minutes. This was proved by means of determining the particle size of 171.1 nm and 173.8±5.89 nm(mean±SD), mean percentage entrapment efficiency 58±3.22 (mean±SD). The in vitro drug profile confirmed that the pH-sensitive liposomes of carboplatin confirmed 26% more drug release in phosphate buffer having pH-5.5 than having pH-7.4 and hence turned into successfully launched in tumor surroundings in which the pH is acidic [28]. The balance research discovered that the optimized components of pH-sensitive liposomes have been extra solid at refrigerated conditions than that at ambient temperature because of their particle length distribution. The significant higher IC₅₀value (42μg/ml)(as determined by MTT assay) of pH-sensitive liposomes as compared to plain carboplatin (21μg/ml) further supported the lower drug release at physiological pH and therefore more drug release at target tumor sites (acidic pH) [29] as shown in Fig. 8. The solid-state characterization studies demonstrated that sucrose was more suitable as a cryoprotectant than lactose for dry powder formulation as Cpt-DPF.

4 Conclusion

It can be concluded that pH-sensitive liposomes designed with carboplatin has been appeared as a promising delivery approach in order to target tumor tissue as evaluated via the pre-scientific studies in each in vitro and ex-vivo situations. The successful pulmonary delivery of Cpt-DPFs counseled their capability use for remedy of pulmonary and systemic sicknesses. Consequently, it has been established that carboplatin loaded pH-sensitive liposomes had been organized efficaciously and their drug release was found to be better at acidic pH (tumor environment) as compared to physiological pH which turned into our prime goal.

The cytotoxicity studies revealed more potential of pH-sensitive liposomes (42μg/ml) as compared to plain carboplatin (21μg/ml) further supported the lower drug release at physiological pH and therefore more drug release at target tumor sites (acidic pH). This preliminary indication of drug release in tumor environment was also supported by way of IC₅₀ of drug loaded pH-sensitive liposomes warrants further clinical studies as the following step in the direction of possible human applications. In the light of above facts, the present work tended to be the advancement of knowledge and it was hoped that these studies would pave way for future research in the area of targeted drug delivery system.

Footnotes

Acknowledgments

Authors acknowledge the laboratory facilities and financial support received from the Department of Pharmaceuticals, Abhilashi College of Pharmacy, and Abhilashi University to carry out the research work.

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Formulation optimization of pH-sensitive liposomes based drug delivery of Carboplatin and anti-proliferative evaluation against A549 (human lung carcinoma) cell lines

Abstract

Keywords

Abbreviations

1 Introduction

2 Experimental section

2.1 Optimization using factorial design

2.1.1 Central composite design

2.1.2 One factor RSM design

2.2 Materials and methods

2.2.1 Chemical substances

2.2.2 Preparation of Calibration curve of carboplatin in PBS (7.4) and in PB (5.5)

2.2.4 Melting point of carboplatin

2.2.5 Apparent partition coefficient of carboplatin

2.2.6 Solubility of carboplatin

2.2.7 Method of preparation

Table 1 Coded values of formulation parameters of carboplatin loaded pH-sensitive liposomes Factors Units Levels used actual (coded) Low (–1) Medium (0) High (1) Drug: Lipid ratio Molar ratio 1:5 1:10 1:15 Volume of hydration media ml 2 3 4

2.2.9 Particle size

2.2.12 Entrapment efficiency: Separation of loose drug from drug entrapped in Liposomes

2.2.13 Evaluation of entrapped drug in liposome

2.2.14 In vitro drug release studies

2.2.15 Solid state characterization

2.2.16 Stability studies

2.3 Biological activity: In vitro cytotoxicity studies

2.3.1 Cell culture and maintenance

2.3.2 Cell viability assay

3 Results and discussion

3.1 Optimization of pH-sensitive liposomes

4 Conclusion

Footnotes

Acknowledgments

References

Table 1
Coded values of formulation parameters of carboplatin loaded pH-sensitive liposomes

Factors Units Levels used actual (coded)

Low (–1) Medium (0) High (1)

Drug: Lipid ratio Molar ratio 1:5 1:10 1:15

Volume of hydration media ml 2 3 4