Liposomes are used as carriers for drugs and antigens because they can serve the purpose of solubilization, protection, can prolong the drug action and can target the drug to certain targets [2]. An advantage of using particle carriers, such as liposomes, is that drugs can easily become encapsulated, either dissolved in the aqueous phase or in the lipid phase, without the requirement of a covalent linkage between drug and carrier [5]. Several strategies have been proposed, to accomplish site-specific triggered release in tumor tissue. Liposomes can alter favorably the pharmacokinetic profile of the encapsulated species and thus provide selective and prolonged pharmacological effects at these sites of administration [12]. Triggered release by liposome may be performed either extracellularly or intracellularly for the treatment of tumors. Since tumors are often characterized by an acidic microenvironment, liposome with compositions leading to destabilization at acidic pH have been proposed to induce the specific release of the encapsulated anticancer drug, extracellularly in the diseased tissue. At the intracellular level, the design of pH-sensitive liposomes to take advantage of the acidic environment of the far endosomes and lysosomes has been more successful because the pH of those intracellular compartments may be below 5.0 [11]. A key lipid for the fusion of liposomes with the endosomal membrane is the dioleyl phosphatidyl ethanolamine (DOPE), but other components are generally associated with giving liposomes pH-sensitive properties. This includes mildly acidic amphiphiles such as oleic acid and cholesteryl hemisuccinate (CHEMS) [10, 16]. The pH-sensitive liposomes increase the aqueous solubility of the drug and also increase the intracellular delivery of drug at acidic pH (in the early and late endosomal stage) and avoid the lysosomal degradation of the drug as well as reduce the systemic side-effect. New liposomes strategies consists of constructs capable of stimuli-sensitive release, such liposomes are designed to go through structural changes in response to physicochemical stimuli, thus allowing more controlled release of the encapsulated drug. These approaches include the use of pH-sensitive liposomes triggered by characteristics acidic milieu of solid tumours [5]. Taking advantages of the altered pH gradients in tumor extracellular environments and in its intracellular compartments, pH-sensitive liposome have been designed to prove the concepts of specific cancer cell targeting, enhanced cellular internalization, and rapid drug release. Especially promising is the concept of intracellular drug delivery by the pH-sensitive liposomes because it offers an efficient means of overcoming the multidrug resistance, one of the major causes for cancer treatment failures and also increases the intracellular delivery of the drug [2, 19]. Docetaxel (Taxotere®) is a second-generation taxane derived from the needles of the European yew tree, Taxus baccata [6]. The synthesis of Docetaxel starts from 10-deacetylbaccarin III, a non-cytotoxic constituent of European yew tree needles [7]. Currently Taxotere® is marketed by Sanofi Aventis; it is clinically effective against advanced breast, ovarian and non-small cell lung cancer. Docetaxel shows very low water solubility, and presently the only available formulation for clinical use consists of a solution (40 mg/ml) in a vehicle containing high concentration of Tween 80®. This vehicle has been associated with several hypersensitivity reactions and has shown incompatibility with common PVC intravenous administration [8]. A major problem of Tween 80® in Taxotere® includes high rates of allergic and/or immune reactions, severe pain at injection sites, serious and potentially permanent damage to blood vessels at or near the site of injection. However, the severe adverse reaction of this drug is not due to the drug itself, but to the excipient polysorbate 80 used in its formulation. In order to eliminate the Tween 80®-based vehicle and in the attempt to increase the drug solubility, alternative dosage forms have been suggested, including liposomes and cyclodextrins. Therefore there is a need for the development of alternative dosage form of Docetaxel devoid of Polysorbate 80 [7]. Hence, the objective of the present study was to prepare, optimize and characterize pH-sensitive liposome containing docetaxel, further evaluation of selected optimized formulation for in vitro diffusion study and in vitro cell cytotoxicity study so as to overcome the limitations associated with the current drug therapy.

MATERIALS AND METHODS:

Materials

Docetaxel was a gift sample from RPG Life Sciences, Mumbai. Hydrogenated soya phosphatidylcholine (HSPC) and Dioleyl phosphatidylethanolamine (DOPE) was obtained from Lipoid, GmbH, Germany. Cholesterol hemisuccinate (CHEMS) was a gift sample from Sigma-Aldrich, USA. Cholesterol (CHOL) was purchased from Merck, India. Chloroform, Methanol was purchased from S. D. Fine Chemicals. A549 (lung carcinoma) cell line was purchased from NCCS, Pune. All other chemical reagents were commercial products of analytical or reagent grade and were used without further purification.

Preparation of Liposomes by TFH

TFH method was selected for the preparation of liposome due to non-tediousness and feasibility at lab scale compared to other techniques [9]. Also, from the viewpoint of stability, hydrogenated phospholipids Soyaphosphatidylchloline (HSPC) was used in this investigation [10]. Docetaxel loaded liposomes consisting of HSPC, DOPE, CHEMS and CHOL were prepared by TFH technique. Briefly, the lipids and CHOL were dissolved in a mixture of chloroform and methanol (2:1) in a 250 ml round bottom flask in different molar ratios. The solvent was evaporated using the rotary flash evaporator. The thin dry lipid film thus formed was hydrated using HEPES buffer (pH 8.2). The formed liposomal dispersion was sonicated using probe sonicator. Resultant liposomes were centrifuged at 8,000 rpm and 4^oC for 20 minutes using Laboratory Centrifuge. Free drug (as sediment) and liposomes (as supernatant) were separated. Liposomal suspension was then characterized for vesicle size and percent drug entrapment (PDE). The liposomal compositions and process parameters were optimized to achieve maximum drug entrapment and minimum size.

Lyophilization of Docetaxel loaded pH sensitive liposomes

Freeze drying technique was used for stabilization and to prevent the leakage of entrapped docetaxel [14]. Batch of docetaxel loaded Liposomal suspension composed of (Drug: Lipid 1:20), (DOPE: HSPC: CHEMS: CHOL 4.5:1.5:2:2) was freeze dried with different cryoprotectant to preserve the vesicular size and shape, hence the PDE. In selected liposomal suspension of docetaxel, batch containing the total lipids equivalent to 125 mg, 375 mg (3 times) of different cryoprotectant were dissolved. This liposomal dispersion then subjected to two stage of freeze drying, the resultant dispersion was deep freezed at -70°C for 24 hr in Deep Freezer, Amancio Lab, Mumbai, to form dry ice cake. The formed dry ice cake containing vials transferred to the Heto Freeze-dry system (Heto dry, Denmark) and lyophilized at the -70°C and 50 mbar for 18 hr. The porous cake thus formed was sized successively through #200 sieves. PDR of freeze dried liposomes were determined following dehydration-rehydration cycle. The effect of use of different cryoprotectants on particle size and PDR of developed lyophilized liposomal product was studied.

Characterization of Liposomes

Morphology

Morphology of the liposomes was ascertained from photomicrographs taken using Olympus BX 40 microscope at a magnification of 40X before ultrasonication.

Particle size and Zeta Potential

The particle size and zeta potential of liposomes was measured by dynamic light scattering with a Malvern Zetasizer Nano-ZS (Malvern Instruments, Malvern, UK). Each sample was suitably diluted 10 times with double distilled water to avoid multi-scattering phenomena and placed in a small disposable zeta cell. The size analysis of a sample consisted of 12 measurements, and the results are expressed as mean size ±S D. Zeta limits ranged from -200 to +200mV. The electrophoretic mobility (µm/sec) was converted to zeta potential by in-built software using Helmholtz-Smoluchowski equation.

Percentage drug entrapment

For the analysis of entrapped drug in liposomes, the liposomes were centrifuged and supernatant was collected. To find out % Entrapment, 0.1 ml of supernatant was dissolved in required ml of MeOH solution. The absorbance was taken at the wavelength of 229 nm against reagent blank on UV-Visible Spectrophotometer (UV-1700, Shimadzu).

Entrapped Drug

% Drug Entrapment = ------------------------------- x 100

Total drug

Differential Scanning Calorimetry

DSC analysis was carried out using a Differential Scanning Calorimeter (DSC-60, Shimadzu, Japan) at a heating rate of 20°C per minute in the range of 30°C to 300°C under inert nitrogen atmosphere at a flow rate of 40ml/min (4 to 8 milligrams of samples were sealed in standard aluminum pans with lids). DSC thermo grams were recorded for Docetaxel, Drug-lipid mixture and lyophilized Docetaxel encapsulated pH-sensitive liposomes.

Scanning Electron Microscopy

The surface morphology of lyophilizedpH-sensitive liposomes was examined by scanning electron microscopy (JSM-5610LV, JEOL, Japan). Samples were attached to sample stubs using double side carbon tape, and then viewed using an accelerating voltage of 5 kilovolt at the magnification of 1000X and 1500X.

Percent drug retained and particle size of Lyophilized pH sensitive liposomes

Percent drug retained (PDR) is the percentage of drug retained in the liposomes after lyophilization or in stability samples to initially drug entrapped in the liposomal dispersion [14]. 10 mg of powder was rehydrated with 1ml of distilled water with gentle, occasional agitation for 30 minutes. The liposomal dispersion thus obtained was separated from the drug leaked during lyophilization cycle by centrifugation method. The liposomal formulations were rehydrated and diluted with distilled water and analyzed for particle size.

In Vitro Drug Diffusion from Liposomes

In-vitro release studies were carried out using dialysis bag release technique which is widely used to evaluate drug release from macro and nanosized carriers. Studies of docetaxel release from the liposomes were conducted at room temperature in triplicate using a SDi permeable membrane (molecular weight cutoff 10,000 Da, MEMBRA-CEL, Chicago, IL) to separate the donor and receptor media. The receptor medium was filled with 20ml of 10% methanolic buffer solutions to ensure solubility. Liposomes suspension containing drug, equivalent to 1mg of docetaxel, was dispersed in the donor medium (5ml). At predetermined intervals of time, 2ml aliquots were withdrawn from the receptor compartment and in order to maintain the sink condition, fresh buffer solution was used to replenish the receptor compartment. Analysis was carried out immediately after withdrawal. The study was carried out up to 30hrs. Samples were withdrawn after 15 min, 30 min, 1, 1.5, 2, 3, 4, 5, 6, 8, 24, and 30hrs. The study was carried out for plain Docetaxel, Docetaxel loaded pH sensitive liposomes and results obtained were compared.

In Vitro Cytotoxicity Study by MTT Assay

Cell cytotoxicity assay was carried out to measure the ability of the cells to survive and continue to proliferate. The in vitro cytotoxicity of the anticancer drugs and their formulations can be evaluated by using MTT Assay [20, 21, 22]. The cytotoxicity of conventional liposomes and pH sensitive liposomes containing docetaxel against A549 lung cancer cells was determined by using the MTT dye reduction assay, and was compared with that of plain docetaxel. Briefly, 10⁴cells/well in its exponential growth phase was plated in 96-well flat-bottom tissue-culture plates. The cells were incubated at 37 ºC, 5% CO₂ in incubator for 24 h, during which cells were attached and resumed to grow as monolayer. Various dilutions were made to the conventional liposomes, pH sensitive liposomes and the plain drug in the sterile PBS to prepare the 0.1μM, 1.0μM, 2.5μM, 5μM and 10μM concentrations, and were added in triplicate (200 mL each). Control wells were treated with equivalent volumes of docetaxel free media. After 24 hr, the supernatant was removed from well plates. Each well was washed with 100mL of PBS. MTT (1 mg/ml) in culture medium (100 mL each) was added to each well and incubated for 4hrs. The unreduced MTT and medium were then discarded after which was added 200 mL of DMSO to dissolve the MTT formazan crystals. Plates were shaken for 20 min and absorbance was read at 595 nm using the microplate reader (ELISA Reader). Cell viability was determined using the formula in Eq. (1)

Mean Absorbance of Sample

% Viability = --------------------------------------- x 100 ……... (1)

Mean Absorbance of Control

Where, absorbance of sample and control cells represents the amount of formazan determined for cells treated with the different formulations and for control cells, respectively. The IC₅₀ values (i.e., concentration resulting in 50% growth inhibition) of docetaxel were graphically calculated from concentration-effect curves, considering the optical density of the control well as 100%.

RESULTS AND DISCUSSION:

Optimization of Process Parameters

Vacuum, speed of rotation and film formation time

Different liposomal batches were prepared by varying the process parameters like vacuum, rotation speed (rpm), and the time of film formation in order to obtain the thin and uniform film. The formation of thin and uniform film was dependent on vacuum and speed of rotation. Vacuum was increased progressively from 500 to 700 mmHg and speed of rotation was varied between 75 to 125 rpm (table 1).

Table1 Effect of optimization of vacuum speed, speed of rotation and film formation time.

Vacuum (mmHg)	Rotation (rpm)	Time (min)	Quality of film (results are drawn after performing the experiments in triplicate)
500	75	60	Uniform but thick and not complete evaporation of solvents (leaves traces of solvent).
500	100	50	Thin and uniform but not completely dried
500	125	30	Thin and irregular but not completely dried
600	75	60	Uniform and dry but thick
600	100	60	Uniform, dry and thin film
600	125	30	Thin but leaves gap in between and irregular
700	75	60	Thin, dry, irregular (ruptured)
700	100	50	Thin, dry, irregular (ruptured)
700	125	30	Thin, dry, irregular (ruptured)

As the vacuum was increased from 500 to 700 mmHg the film formation time was decreased, but above 600 mmHg the film formed was not uniform. Increase in speed of rotation from 75 to 100 rpm produced dried and uniform film and further increase in speed of rotation beyond 100 to 125 the film was ruptured and non uniform. Optimized conditions for the speed of rotation and vacuum were, initially the vacuum was kept at the 400 mmHg for the complete and smooth uniform film formation. After 5min condition was 600 mmHg, 100 rpm, and 60 minutes.

Composition of solvent (CHCl₃: MeOH)

The film was prepared using different ratio of CHCl₃: MeOH and evaluated to obtain optimum solvent ratio which gives uniform and thin film (table 2).

Table 2 Optimization of solvent ratio

CHCl₃:MeOH	Observations (after performing the experiments in triplicate)
1:1	No proper film, not proper hydration
2:1	suitable
1:2	No proper film, not proper hydration
4:1	No proper film, not proper hydration

The solvent system composition should be such that it prevents precipitation of formulation components during solvent stripping process. The different composition of organic solvent systems of CHCl₃: MeOH (1:1, 2:1, 1:2 and 4:1) were used for dissolving the formulation components like HSPC, DOPE, CHEMS, CHOL, and drug and evaluated to obtain optimum solvent ratio which gives uniform film. The results reveal that uniform film and proper hydration was obtained with a CHCl₃: MeOH ratio of 2:1.

Hydration Volume

The film was hydrated using different hydration volume and evaluated to obtain optimum hydration volume which gives complete hydration of lipid film with maximum entrapment and uniform size (table 3). The optimum volume of hydration medium is required to ensure complete hydration of the planar bilayers to form the spherical liposomes.

Table 3 Optimization of hydration volume

Hydration Volume	Observations (after performing the experiments in triplicate)
1	Not proper hydration
2	Uniform liposome and acceptable PDE
3	No further improvement

For pH-sensitive liposomes, 2ml of hydration volume was found optimal. Docetaxel being lipophilic drug, 2ml of hydration gave uniform liposomes and acceptable PDE.

Hydration time

Film was hydrated for different time intervals between 30 min, 60 min, and 90 min in order to find out the time of hydration which gives complete hydration of lipid film with maximum entrapment and uniform size (table 4).

Table 4 Optimization of Hydration Time

Hydration time	Effect on hydration of lipid film
30 min	Not properly hydrated leaves film behind.
60 min	Complete hydration and optimum entrapment efficiency.
90 min	No significant effect on entrapment efficiency.

The results revealed that after 30 minutes the film was not properly hydrated, some portion of lipid was still remained unhydrated on the surface of the RBF; at 60 minutes the film was completely hydrated and gives homogenous suspension of liposome with optimum entrapment efficiency and beyond 60 min there was a no significant increase in PDE but the unhydrated lipids were come out which interfere in particle size evaluation.

Sonication cycles

Prepared liposomal dispersion are subjected to sonication to get the nano sized liposomal suspension for the parenteral delivery using probe sonicator (80 % amp, 0.6 cycle and 30 seconds for each cycle) and evaluated for % entrapment and liposomes size (table 5) at different cycles (1, 2 and 3 cycles; one cycle is 30 sec).

Table 5 Optimization of Sonication Cycle.

No. of Cycles	Liposomes
No. of Cycles	PDE*	Vesicles Size*
1	71.45±1.456	276.4 nm ± 2.197
2	69.24±0.703	166.5 nm ± 1.259
3	62.36±0.729	134.5 nm ± 0.483
4	59.26±1.983	110.3 nm ±1.831

*Values are Mean ±SD (n= 3)

Results indicate that after 3 cycles the liposomal vesicles were achieved with satisfactory PDE and size. Hence, optimized sonication cycle selected was 3 cycles (80% amp, 0.6 cycles).

Optimization of formulation parameters

Optimization of drug: Lipid ratio for maximum entrapment efficiency

Batches with different Drug: lipid ratios were prepared and entrapment efficiency was determined (table 6).

Table 6 Optimization of Drug: Lipid ratio

SR NO	DRUG:LIPID	LIPID:CHEM: CHOL	HSPC: DOPE (LIPID)	PDE*
1	1:3	6:2:2	1:1	Flaking
2	1:5			Flaking
3	1:10			46.53± 1.832
4	1:15			59.67± 0.574
5	1:20			69.36± 0.729
6	1:25			71.49± 1.351

* Values are Mean± SD (n=3)

Increase in the lipid proportion relative to drug led to the increase in the drug entrapment from 46.53±1.832% (1:10) to 69.36±0.729 (1:20). With increase in quantity of lipids the more numbers of liposomes vesicles were formed per ml of the liposomal dispersion resulting in increased drug entrapment [10, 16]. But the proportionate increase in % drug entrapment is compensated with proportionate increase in lipids i.e. to use more lipids to entrap constant amount of drug. Hence, drug: lipid ratio of 1:20 was selected due to acceptable drug entrapment of docetaxel.

Optimization of hydration media

Optimization of hydration media was carried out with respect to entrapment efficiency of liposomes. Different batches of liposomes were prepared using different hydration media such as distilled water, PBS pH 7.4, and HEPES buffer pH 8.2 but keeping constant hydration volume and hydration time (table 7)

Table 7 Optimization of hydration media for liposome

Sr no.	Drug: Lipid	Lipid: CHEMS: CHOL	Hydrating media	PDE*
1	1:20	6:2:2	Distilled water	22.48±1.562
2			PBS pH 7.4	69.36±0.729
3			HEPES buffer pH 8.2	72.43±1.267

*Values are Mean±SD (No. 3)

Optimization of Lipid: Cholesterol ratio

An effect of lipid (HSPC, DOPE and CHEMS) to cholesterol ratio was evaluated with respect to entrapment efficiency. Different batches of liposomes were prepared using different Lipid: Cholesterol ratio but keeping constant Drug: lipid ratio (table 8).

Table 8 Optimization of Lipid to Cholesterol ratio of liposomes

Sr no	Drug: Lipid	Lipid:CHEMS:CHOL	Lipid (HSPC:DOPE)	PDE*
1	1:20	6:2:2	1:1	72.43±1.267
2		6.5:2:1.5		67.78±2.453
3		7:2:1		63.64±1.304

* Values are Mean±SD (n=3)

Entrapment efficiency of liposomes prepared using different LIPID: CHOLESTEROL ratio was determined and results shows that at 8:2 lipid: cholesterol ratio gives maximum PDE value. Cholesterol concentration is important because it gives the stability (rigidity) to the liposomal vesicles which provide stability during their circulation in blood. Cholesterol also alters the permeability and fluidity of the bilayer and prevents the major leakage of the entrapped drug from the liposomes, thus improve the PDE [12].

Optimization of CHEMS ratio

Optimization of CHEMS was carried out with respect to entrapment efficiency of liposomes. Different batches of liposomes were prepared using different molar ratio of CHEMS. Entrapment efficiency was determined (table 9).

Table 9 Optimization of CHEMS ratio of liposomes

SR NO	DRUG:LIPID	LIPID (HSPC: DOPE 1:1)	CHEMS	CHOL	PDE*
1	1:20	8	0	2	Incomplete hydration
2		7	1	2	60.45±1.053
3		6	2	2	72.43±1.267

* Values are Mean±SD (n=3)

Entrapment efficiency of liposome prepared using different Lipid: CHEMS ratio was determined and results shows that in absence of CHEMS incomplete hydration occurs but increase in CHEMS causes increase in entrapment efficiency since it facilitate and stabilize the bilayer formation of DOPE. DOPE because of its small head group not able to form bilayer structure. Therefore acidic amphiphiles such as CHEMS was added that act as stabilizer and facilitate the bilayer formation of DOPE at alkaline or neutral pH. At alkaline or neutral pH, however, the negatively charged amphiphiles will reduce the intermolecular repulsion of DOPE head groups and stabilize the structure in bilayer organization. The pK value of carboxylic acid of CHEMS is 5.8, the carboxylic acid would be ionized above a pH, such as pH 6, 7, 8, and so that head group is large enough to stabilize DOPE bilayers. On the other hand, when the pH was below the pK value, such as pH 5, the carboxylic acid would be deionized and thus the size of the head group of CHEMS decreases. Accordingly the DOPE bilayer would be destabilized into non-bilayer structure, giving rise to higher release. Optimized Lipid: CHEMS ratio for pH-sensitive liposome selected was: 8:2

Optimization of HSPC: DOPE Ratio

Optimization of HSPC: DOPE was carried out with respect to entrapment efficiency, particle size and zeta potential of liposomes and further conformation is done on the basis of pH-sensitivity of liposomes. Different batches of liposomes were prepared using different molar ratio of HSPC: DOPE (table 10).

Table 10 Optimization of HSPC: DOPE ratio of liposomes

Sr no	LIPID:CHEM:CHOL	HSPC: DOPE (LIPID)	PDE*	PARTICLE SIZE (PDI)*	ZETA POTENTIAL
1	6:2:2	1:1	72.43±1.267	123.1±2.147 (0.256)	-61.3
2		1:2	68.39±1.185	129.1±3.672 (0.266)	-45.6
3		1:3	64.79± 0.230	111.0±2.713 (0.227)	-22.8

* Values are Mean±SD, (n=3)

DOPE concentration is important since it is mainly responsible for inducing the pH-sensitivity to the liposome. So the main optimization of HSPC: DOPE ratio is done on the basis of the pH-sensitivity of the liposome.

Various process (table 11) and formulation (table 12) parameters were optimized, to get maximum loading of drug into liposomes and minimum size.

Table 11 Optimized process parameters

Parameters	Optimized Value
Composition of solvent system ( CHCl₃:MeOH )	2:1
Vacuum applied	600mmHg (400 mmHg First 15 min.)
Solvent evaporation time	60 min
Speed of rotation	100 rpm/70 rpm
Hydration time	60 min
Hydration volume	2 ml
No. of sonication cycles 30 sec	3 cycles ( 80% amp, 0.6 cycle)

Table 12 Optimized formulation parameters:-

Parameters	Optimized value
Drug: Lipid	1:20
Hydration media	HEPES buffer pH 8.2
LIPID: CHOL	8:2
LIDIP: CHOL: CHEMS	6:2:2
HSPC: DOPE	1:3

Characterization

Morphology A bilayer vesicular system is being seen (fig. 1).

Figure 1 Morphology of liposomes

Particle size and Zeta potential

Particle size (fig. 2) and zeta potential (fig.3) of optimized pH-sensitive liposomal formulation was found to be 111.0±2.713nm with PDI of 0.227 and -22.8 mV (fig.3), attributed to the anionic nature of lipid. Zeta potential value (-30 to +30 mV) increases the stability of liposome by avoiding particle aggregation.

Figure 2 Particle size of pH-sensitive Liposomes: 111.0 nm (PDI: 0.227)

Figure 3 Zeta potential of pH-sensitive liposomes: -22.8 mV

Percentage entrapment efficiency

Percentage entrapment efficiency for optimized pH-sensitive liposomal formulation was 64.79±0.230. Higher percentage entrapment efficiency can be attributed to the lipophilic nature of drug.

DSC Study

DSC is useful in the investigation of the thermal properties of drug delivery carriers, providing both qualitative and quantitative information about the physicochemical state of drug inside the drug delivery systems. There is no detectable endotherm if the drug is present in a molecular dispersion or solid solution state in the polymeric systems loaded with drug. In the present investigation, DSC of pure docetaxel and docetaxel encapsulated liposomes were carried out. DSC thermogram of Docetaxel shows the endothermic peak around 225ºC which is slightly different from the reported value i.e. 192ºC. DSC thermogram of lyophilized pH -sensitive liposome showed no related peak for docetaxel indicating that docetaxel in the liposome was in an amorphous or disordered-crystalline phase of molecular dispersion or a solid solution state in the lipid layer of liposome. DSC thermograms of pure drug (Docetaxel) (fig. 4), Lipid mixture (fig. 5) and lyophilized formulation (fig. 6) are presented below.

Figure 4 DSC thermogram of docetaxel.

Figure 5 DSC thermogram of lipid mixture

Figure 6 DSC thermogram of lyophilized pH -sensitive liposome

SEM Study

SEM imaging of docetaxel loaded liposome exhibit a spherical shape of liposome (fig.7).

Figure 7 SEM image of Docetaxel loaded pH-sensitive liposomes

Percent drug retained and particle size of Lyophilized pH sensitive liposomes

Different sugars have markedly different effect on stability. The apparent difference between the ability of these sugars, amino acids and polyols to preserve dry liposomes may be related to fundamental difference in their mode of interaction with the bilayer [14, 15]. Sucrose and Trehalose were the effective cryoprotectant, as they provide dry free flowing powder of the liposomal formulation with easily resuspendable formulation. Batch PL₂containing 3 times Trehalose to the total lipid weight show highest PDR (92.76±0.571) (table 13) and particle size (173.4±3.721) (figure 8), compared to all other prepared formulations.

Table 13 Influence of Cryoprotectants on freeze dried Liposomes

Batch Name	Cryoprotectant	Rehydrated liposome
Batch Name	Cryoprotectant	PDR (%)*	Particle Size (nm)*
PL1	Sucrose (3 times)	91.71±0.283	198.1±9.253
PL₂	Trehalose (3 times)	92.76±0.571	173.4±3.721
PL₃	Mannitol (3 times)	90.93±0.414	214.2±8.142
*Mean±SD (No. 3)

Figure 8 Particle size of pH-sensitive liposomes after lyophillization

In Vitro Drug Diffusion from Liposomes

Comparative In-Vitro diffusion studies were carried up to 30 hrs for pH-sensitive liposomes, plain docetaxel solution and conventional liposomes (table 14 and figure 9 and 10). The release of docetaxel from pH-sensitive liposomes in PBS buffer medium from pH 5.0 and pH 7.4 were investigated. The release rate of docetaxel in the media with different pH values was different. The release showed pH-dependent. The release rate of docetaxel at neutral or alkaline pH was slow and sustained. However in the weak acidic environment surrounding the tumor releases were much faster. It was found that the pH value of dissolution medium affect the drug release rate of docetaxel. The drug release rate reduced with the increasing of the pH value of dissolution medium.

In Vitro Cytotoxicity Study by MTT Assay

Cell cytotoxicity study by MTT assay for Docetaxel, pH-sensitive liposome and Conventional liposome were investigated against A549 cell. For A549 cells, after 48 hrs incubation, pH-sensitive liposome (IC₅₀=6.1µM) exhibit superior cytotoxic activities to conventional liposome (IC₅₀=7.8µM) and free docetaxel (IC₅₀=9.6µM). This indicate that pH-sensitivity of liposome played an important role in enhancing cytotoxic effect by increasing the intracellular release of the drug. Free docetaxel shows less cytotoxicity compared to conventional and pH-sensitive liposome, resulting from the reduced cellular uptake of docetaxel. For free docetaxel, a multi-drug resistant effect, out-fluxing docetaxel through the p-glycoprotein pump, might play an additional role in decreasing the intracellular concentration of docetaxel. Therefore, increased intracellular release due to destabilization of liposomal membrane at endosomal pH may be mainly responsible for the higher cytotoxic effect. The cell viability of docetaxel and its formulations at different concentrations (µM) has been presented in (table 15). With increase in concentration, % cell viability is decreasing.

Time (hrs.)	Plain drug % drug diffused (Mean±SD)		Time (hrs.)	Conventional liposome (HSPC:CHEMS:CHOL 6:2:2)		pH-sensitive liposome (HSPC: DOPE: CHEMS: CHOL 1.5:4.5:2:2)
	% drug diffused (Mean±SD)			% drug diffused (Mean±SD)
	pH 7.4	pH 5.0		pH 7.4	pH 5.0	pH 7.4	pH 5.0
0.25	6.33±1.983	5.46±1.319	0.5	2.31±0.274	1.34±2.331	1.15±1.032	4.63±1.203
0.50	21.33±1.395	24.31±0.291	1	6.82±1.495	5.98±1.726	4.28±0.927	10.42±2.318
0.75	39.19±1.290	42.49±0.923	1.5	8.54±1.578	9.47±1.483	7.84±1.841	23.81±1.276
1.0	45.19±2.164	54.26±1.623	2	12.31±1.439	15.36±2.197	15.63±1.760	34.59±1.471
1.5	64.96±1.223	60.42±1.342	3	15.3±0.327	16.74±1.583	18.60±1.921	42.36±1.203
2.0	75.93±1.092	69.31±1.287	4	24.65±1.648	27.54±1.721	26.51±1.823	57.79±1.627
3.0	87.43±1.487	79.02±1.739	6	30.67±1.975	31.76±1.429	32.54±1.276	76.57±1.492
4.0	94.21±1.496	89.73±1.695	8	35.86±1.540	34.74±1.361	45.42±1.672	82.96±1.721
5.0	97.86±1.267	95.62±1.426	24	79.56±1.684	81.32±1.644	81.83±1.296	87.31±1.934
-	-	-	30	86.45±1.497	89.32±1.927	88.23±1.745	91.07±1.429

Concentration (µM)	Cell viability (%)
Concentration (µM)	Docetaxel	Conventional liposome	pH-sensitive liposome
0.1	98.45	97.10	95.22
1.0	94.24	96.09	83.41
2.5	78.03	79.54	67.96
5.0	69.42	61.66	54.35
10.0	48.96	41.63	38.29

Formulation	IC₅₀ value (µM)
Docetaxel	9.6
Conventional liposome	7.8
pH-sensitive liposome	6.1