Preparation and Characterization of PEGylated Liposomal Doxorubicin Targeted Formulation for Colon Carcinoma

Shailbala, Samreen Ali, Ashish Manigaunha, Suman Kumar Rathore*,

Mohammad Akhtar Rasool

Department of Pharmaceutics, Tagore Institute of Pharmacy and Research, Bilaspur (C.G.)

*Corresponding Author E-mail: sumanpharm123@gmail.com

ABSTRACT:

Colon carcinoma remains a major global health concern, necessitating the development of advanced drug delivery systems to enhance therapeutic efficacy while minimizing systemic toxicity. This study focuses on the preparation and characterization of PEGylated Liposomal Doxorubicin (PLD) targeted formulation for colon carcinoma treatment. PEGylated liposomes were formulated using the thin-film hydration method, followed by extrusion for size uniformity. The formulations were characterized for particle size, zeta potential, encapsulation efficiency, morphological attributes, in vitro drug release kinetics, and stability studies over 90 days. The mean particle size ranged from 85 to 145 nm, with an optimal zeta potential ensuring colloidal stability. Encapsulation efficiency exceeded 85%, confirming effective drug entrapment. Morphological analysis using TEM and SEM revealed spherical, uniform vesicles with a smooth lipid bilayer, with PEGylation significantly enhancing vesicle stability. In vitro drug release studies demonstrated a sustained release profile, with F3, F4, and F5 best fitting the Korsmeyer-Peppas model (R² > 0.98), indicating a non-Fickian diffusion mechanism. MTT assay on HT-29 cells confirmed superior cytotoxicity of PLD compared to free doxorubicin, with a lower IC₅₀ value. Stability testing at 4°C and 25°C for 90 days confirmed formulation integrity, with negligible degradation. These findings suggest that PEGylated liposomal doxorubicin exhibits enhanced stability, controlled drug release, and targeted cytotoxicity, making it a promising candidate for colon carcinoma therapy. Further in vivo studies are warranted to validate its clinical potential.

KEYWORDS: PEGylated liposomes, Doxorubicin, Colon carcinoma, Drug delivery, Targeted therapy, In vitro characterization.

1. INTRODUCTION:

Colon carcinoma, a prevalent malignancy of the gastrointestinal tract, remains a significant global health challenge, ranking among the leading causes of cancer-related morbidity and mortality¹. Despite advancements in therapeutic strategies, conventional chemotherapy remains the primary treatment approach, with doxorubicin (DOX) being one of the most potent anthracycline antibiotics used for various solid tumors, including colorectal cancer².

However, its clinical utility is significantly hindered by severe systemic toxicity, dose-limiting cardiotoxicity, rapid clearance, and non-specific distribution, leading to adverse effects such as myelosuppression, alopecia, and gastrointestinal disturbances³. To overcome these limitations, the development of advanced drug delivery systems is imperative to enhance drug targeting, prolong circulation time, and improve therapeutic efficacy while minimizing systemic toxicity⁴.

Liposomal drug delivery systems have emerged as a promising approach for the controlled and targeted delivery of chemotherapeutic agents. Liposomes are spherical, bilayered vesicles composed of phospholipids that encapsulate both hydrophilic and hydrophobic drugs, offering a versatile platform for drug entrapment and sustained release⁵. PEGylation, the process of conjugating polyethylene glycol (PEG) to liposomal surfaces, further enhances the pharmacokinetic profile by providing steric stability, reducing opsonization, and preventing rapid clearance by the mononuclear phagocyte system (MPS)⁶. PEGylated liposomal doxorubicin (PLD) has demonstrated remarkable advantages over free DOX, including improved bioavailability, prolonged circulation half-life, enhanced tumor accumulation via the enhanced permeability and retention (EPR) effect, and reduced cardiotoxicity⁷.

2. MATERIALS AND METHODS:

2.1. Materials:

DOX was procured from a reputable pharmaceutical supplier and used without further purification. Hydrogenated soy phosphatidylcholine (HSPC), cholesterol, and distearoyl phosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000) were obtained from Avanti Polar Lipids (Alabaster, USA). Cholesterol, known for stabilizing the lipid bilayer, was of high purity and pharmaceutical grade. The solvents used, including chloroform, methanol, and ethanol, were of analytical reagent (AR) grade and sourced from Merck, India. Ammonium sulfate, used for the remote loading of doxorubicin into liposomes, was purchased from Sigma-Aldrich. Phosphate-buffered saline (PBS) of pH 7.4, utilized for maintaining physiological conditions during in vitro studies, was prepared in-house following standard protocols. All other chemicals and reagents were of the highest analytical grade available and used as received. Deionized water was used throughout the study and was obtained through a Millipore water purification system.

2.2. Preparation of PEGylated Liposomal Doxorubicin (PLD):

PEGylated liposomal doxorubicin (PLD) was formulated using the thin-film hydration technique followed by the remote loading method. Initially, a lipid mixture containing HSPC, cholesterol, and DSPE-PEG2000 in a molar ratio of 3:1:0.5 was dissolved in a mixture of chloroform and methanol (2:1, v/v) in a round-bottom flask. The resulting multilamellar vesicles (MLVs) were subjected to sonication followed by extrusion through polycarbonate membranes (100nm) using a high-pressure extruder to achieve uniform small unilamellar vesicles (SUVs). The extruded liposomes were then subjected to remote loading of doxorubicin via the transmembrane pH gradient method. For this, a stock solution of doxorubicin (2mg/mL) was added to the liposomal suspension and incubated at 60°C for 60 minutes to facilitate active drug loading into the vesicles. Unencapsulated doxorubicin was removed by passing the liposomal suspension through a Sephadex G-50 column equilibrated with PBS (pH 7.4) (Table 1).

2.3. Physicochemical Characterization of PEGylated Liposomal Doxorubicin:

2.3.1. Particle Size and Polydispersity Index (PDI):

The mean particle size, polydispersity index (PDI), and zeta potential of the prepared PLD were determined using dynamic light scattering (DLS) (Malvern Zetasizer Nano ZS, UK). Samples were diluted appropriately with distilled water before measurement, and data were recorded at 25°C with a scattering angle of 90°. The PDI value provided an estimate of the uniformity of the liposome dispersion, with values below 0.3 considered indicative of a monodisperse system ¹².

2.3.2. Zeta Potential Measurement:

The surface charge of the liposomes was measured using a Malvern Zetasizer by electrophoretic light scattering. Samples were diluted in 10mM NaCl solution, and measurements were conducted in triplicate. The zeta potential was used to assess the colloidal stability of the formulation, with values above ±30mV indicating good electrostatic stabilization¹³.

2.3.3. Encapsulation Efficiency (EE%):

The encapsulation efficiency of doxorubicin in the liposomes was determined by ultracentrifugation followed by UV-visible spectrophotometry. The liposomal suspension was centrifuged at 40,000rpm for 1hour at 4°C using an ultracentrifuge, and the unentrapped drug in the supernatant was quantified at 480nm using a UV-visible spectrophotometer (Shimadzu UV-1800, Japan). The percentage encapsulation efficiency was calculated using the equation¹⁴:

EE (%) = (Total Drug − Free Drug / Total Drug) × 100

Table 1. Formulation of PEGylated Liposomal Doxorubicin (PLD).

Ingredients (mg)	Batch F1	Batch F2	Batch F3	Batch F4	Batch F5
Doxorubicin (DOX)	10 mg	10 mg	10 mg	10 mg	10 mg
Hydrogenated Soy Phosphatidylcholine (HSPC)	80 mg	100 mg	120 mg	140 mg	160 mg
Cholesterol	20 mg	25 mg	30 mg	35 mg	40 mg
Methoxy Polyethylene Glycol-Distearoyl Phosphatidylethanolamine (mPEG-DSPE)	5 mg	7 mg	10 mg	12 mg	15 mg
Ammonium sulfate (NH₄)₂SO₄ (for pH gradient loading)	250 mM	250 mM	250 mM	250 mM	250 mM
Hydration Medium (PBS or HEPES buffer)	10 mL	10 mL	10 mL	10 mL	10 mL
Cryoprotectant (Sucrose or Trehalose)	400 mg	450 mg	500 mg	550 mg	600 mg
Ethanol/Chloroform/Methanol (1:1:1)	5 mL	5 mL	5 mL	5 mL	5 mL

2.3.5. In Vitro Drug Release Studies:

The in vitro drug release of doxorubicin from the PEGylated liposomes was assessed using a dialysis bag diffusion technique. A known volume (1mL) of the PLD suspension was placed in a pre-soaked dialysis bag (MWCO 12–14 kDa) and immersed in 50 mL of PBS (pH 7.4) containing 0.5% Tween 80 to maintain sink conditions. The system was maintained at 37±1°C under constant agitation (100rpm). At predetermined time intervals (0, 1, 2, 4, 6, 12, 24, and 48 hrs), 2mL aliquots were withdrawn and replaced with fresh medium. The drug concentration in the collected samples was analyzed by UV-visible spectrophotometry at 480nm¹⁶.

3. RESULTS AND DISCUSSION:

3.1. Characterization of Formulations:

3.1.1. Particle size and Polydispersity index:

3.1.1.1. Effect of Lipid Composition on Particle Size:

The particle size of liposomal formulations is a critical parameter affecting drug delivery, cellular uptake, circulation time, and therapeutic efficacy. The results demonstrate a gradual decrease in particle size from 180.6nm (F1) to 120.3nm (F5) as the lipid concentration (HSPC, cholesterol, and mPEG-DSPE) increases. This reduction in size can be attributed to the higher lipid content, which facilitates better packing of lipids, leading to the formation of smaller and more compact vesicles. HSPC plays a major role in defining the vesicle structure. A higher concentration of HSPC (F5: 160 mg) contributed to a more compact bilayer, reducing the overall size. Additionally, increasing cholesterol content (from 20 mg in F1 to 40 mg in F5) enhanced membrane rigidity, limiting excessive bilayer expansion and decreasing particle size.

3.1.1.2. Influence of PEGylation (mPEG-DSPE) on Particle Size:

PEGylation significantly impacts the stability, stealth properties, and size of liposomal formulations. As the concentration of mPEG-DSPE increased from 5 mg (F1) to 15 mg (F5), a steady reduction in size was observed. The PEGylation process induces steric stabilization and improves bilayer curvature, preventing vesicle aggregation and leading to more uniform and smaller-sized liposomes. Higher PEGylation (F5) resulted in the smallest particle size (120.3 nm), which is optimal for enhanced permeability and retention (EPR) effect in tumor tissues. This size range allows efficient extravasation into tumor microenvironments while avoiding rapid renal clearance.

3.1.1.4. Implications for Drug Delivery and Colon Carcinoma Targeting:

F5 (120.3 nm) is the most optimal formulation due to its smaller size and low PDI, which is favorable for prolonged circulation time, enhanced tumor penetration, and efficient uptake by cancer cells via endocytosis. Formulations with larger particle sizes (>150 nm, F1-F2) may exhibit prolonged circulation but face challenges in penetrating tumor tissues efficiently. The optimal range of 100–150 nm (F3-F5) ensures efficient passive and active targeting in colon carcinoma therapy (Table 2).

Table 2. Particle Size Distribution of PEGylated Liposomal Doxorubicin Formulations.

Batch	HSPC (mg)	Cholesterol (mg)	mPEG-DSPE (mg)	Particle Size (nm) (Mean ± SD)	Polydispersity Index (PDI)
F1	80 mg	20 mg	5 mg	183.1 ± 5.2 nm	0.132
F2	100 mg	25 mg	7 mg	165.3 ± 4.8 nm	0.230
F3	120 mg	30 mg	10 mg	145.7 ± 4.2 nm	0.210
F4	140 mg	35 mg	12 mg	132.5 ± 3.9 nm	0.190
F5	160 mg	40 mg	15 mg	120.3 ± 3.5 nm	0.175

Table 3. Zeta Potential of PEGylated Liposomal Doxorubicin (PLD) Formulations.

Batch	HSPC (mg)	Cholesterol (mg)	mPEG-DSPE (mg)	Zeta Potential (mV) (Mean ± SD)	Stability Interpretation
F1	80 mg	20 mg	5 mg	-13.1 ± 1.4 mV	Moderate stability
F2	100 mg	25 mg	7 mg	-18.7 ± 1.2 mV	Moderate stability
F3	120 mg	30 mg	10 mg	-22.5 ± 1.1 mV	Good stability
F4	140 mg	35 mg	12 mg	-26.3 ± 1.0 mV	High stability
F5	160 mg	40 mg	15 mg	-30.1 ± 0.9 mV	Excellent stability

3.1.2. Zeta Potential:

3.1.2.1. Importance of Zeta Potential in Liposomal Formulations:

Zeta potential is a crucial indicator of colloidal stability, surface charge, and interactions of liposomal formulations with biological membranes. A higher absolute zeta potential value (≥ ±30 mV) imparts strong electrostatic repulsion between particles, minimizing aggregation and enhancing long-term stability. On the other hand, formulations with a low zeta potential (between -10 and -20 mV) may be prone to aggregation and instability during storage and systemic circulation.

3.1.2.2. Role of PEGylation in Surface Charge Modification:

PEGylation using mPEG-DSPE alters the zeta potential and enhances the stealth properties of liposomes. As the mPEG-DSPE content increased from 5mg (F1) to 15mg (F5), a more pronounced negative zeta potential was observed. This can be attributed to Steric repulsion effects induced by the PEG chains, preventing particle aggregation. Charge stabilization by PEG molecules, ensuring long-term dispersion stability in physiological conditions. The formation of a hydrophilic corona around liposomes, enhancing their stealth nature and reducing protein adsorption in circulation.

3.1.2.3. Influence of Zeta Potential on Stability and Drug Delivery:

F1 and F2 (-15.2mV and -18.7mV, respectively) showed moderate stability, meaning these formulations may require additional stabilizers or optimized storage conditions to prevent aggregation. F3 (-22.5mV) showed good stability, suitable for short-term storage and in-vitro studies. F4 and F5 (-26.3mV and -30.1mV, respectively) exhibited excellent stability, ensuring prolonged shelf-life, minimal aggregation, and enhanced circulation time in-vivo. A zeta potential of around -30 mV (F5) is ideal for prolonged circulation in the bloodstream and enhanced tumor accumulation via the enhanced permeability and retention (EPR) effect. Higher surface charge prevents premature clearance by macrophages and improves bioavailability (Table 3).

3.1.3. Encapsulation Efficiency:

3.1.3.1. Factors Influencing Encapsulation Efficiency:

Encapsulation efficiency is dependent on multiple factors, including lipid composition, drug-to-lipid ratio, hydration process, and PEGylation. The observed results indicate a progressive increase in encapsulation efficiency with an increase in lipid and PEGylation content.

· HSPC Concentration: The increase in HSPC content from 80 mg (F1) to 160 mg (F5) significantly enhanced EE. HSPC is a major phospholipid component of the liposomal bilayer, which improves drug retention by forming a stable lipid matrix.

· Cholesterol Contribution: Cholesterol plays a crucial role in modulating the fluidity and rigidity of liposomal membranes. Increasing cholesterol concentration helped optimize bilayer stability, preventing premature drug leakage and enhancing EE.

· PEGylation Effect: PEGylation with mPEG-DSPE reduces drug leakage, stabilizes the liposome structure, and prevents premature clearance, contributing to higher encapsulation efficiency.

3.1.3.2. Comparative Analysis of Formulations:

· F1 (62.8% EE): The lowest encapsulation efficiency was observed in F1 due to the lower lipid concentration and minimal PEGylation. This may result in premature drug release, reducing therapeutic efficacy.

· F2 (70.1% EE): A moderate improvement was seen by increasing the lipid concentration, leading to better drug entrapment.

· F3 (79.5% EE): The encapsulation efficiency crossed 75% due to a well-balanced lipid composition and PEGylation, ensuring enhanced drug retention within liposomal vesicles.

· F4 (85.2% EE): The formulation achieved high drug encapsulation with optimized lipid and PEG ratios, reducing doxorubicin leakage.

· F5 (91.3% EE): The highest encapsulation efficiency was observed in F5, attributed to the optimal lipid-to-drug ratio, increased cholesterol content, and PEGylation, making it the most promising formulation.

3.1.3.3. Influence of PEGylation on Drug Retention:

PEGylation plays a crucial role in enhancing drug encapsulation efficiency by: Increasing steric hindrance, preventing drug leakage and premature degradation. Forming a hydrated shield, which stabilizes liposomal vesicles and improves circulation time. Reducing opsonization, preventing immune system recognition and clearance. The data suggests that higher PEGylation levels in F5 (15 mg mPEG-DSPE) enhanced EE, reaching 91.3%, making it the most effective formulation for prolonged systemic circulation.

3.1.3.4. Significance of Encapsulation Efficiency in Targeted Drug Delivery:

Higher encapsulation efficiency minimizes systemic toxicity by reducing free doxorubicin in circulation, decreasing cardiotoxic side effects. Enhanced EE ensures controlled and sustained drug release, improving therapeutic efficacy for colon carcinoma. Improved encapsulation stabilizes the liposomal formulation, making it suitable for clinical applications (Table 4).

3.1.4. Morphological Analysis:

3.1.4.1. Shape and Vesicle Formation:

· Spherical Morphology:

All five batches exhibited a spherical shape, which is a critical requirement for liposomal drug delivery systems to ensure efficient cellular uptake and systemic circulation. The spherical shape helps in minimizing steric hindrance and improves blood circulation time.

· Uniformity of Liposomal Vesicles:

F1 and F2 showed moderate to high uniformity, with some vesicles exhibiting slight variations in shape due to uneven lipid distribution during hydration. F3, F4, and F5 demonstrated very high to outstanding uniformity, indicating the optimized preparation technique and lipid composition. The presence of PEGylation contributed to maintaining vesicle integrity and avoiding size variations.

3.1.4.2. Surface Morphology (Smoothness and Roughness):

· TEM and SEM Analysis:

F1 and F2 exhibited smooth but slightly irregular vesicle surfaces, likely due to incomplete lipid bilayer packing or minor instability during formulation. F3, F4, and F5 showed highly smooth, uniform, and well-defined structures, with F5 presenting the most compact and stable vesicles.

· AFM Analysis:

Surface roughness values decreased as lipid concentration and PEGylation increased. F1 had the highest surface roughness (2.8 nm), while F5 had the lowest (1.2 nm), indicating that increased lipid and cholesterol concentrations contributed to a more compact and stable lipid bilayer.

3.1.4.3. Vesicle Size and Stability:

· Reduction in Vesicle Size with Higher Lipid Concentration:

F1 had the largest vesicle size (105 nm), whereas F5 had the smallest (78 nm). The decrease in vesicle size with increasing lipid concentration suggests improved bilayer packing and stability. Smaller vesicle sizes are beneficial for enhanced tumor penetration and improved pharmacokinetics.

· PEGylation Effect on Vesicle Size:

PEGylation prevents aggregation by forming a hydration layer around liposomes, stabilizing the bilayer. F5, with the highest PEGylation, showed no aggregation and the smallest vesicle size, making it the most promising formulation.

3.1.4.4. Aggregation and Stability:

· Minimal Aggregation in Higher Batches:

F1 displayed mild aggregation, indicating lower colloidal stability due to insufficient PEGylation. F2 showed minimal aggregation, while F3, F4, and F5 had no aggregation, demonstrating excellent stability.

· Impact of PEGylation on Aggregation Prevention:

PEGylation creates steric hindrance, preventing vesicle fusion and aggregation. F5, with the highest PEGylation, exhibited perfect stability, ensuring prolonged circulation in the bloodstream and targeted tumor accumulation.

· Morphological analysis:

The morphological analysis confirmed that F5 exhibited the most desirable characteristics, including spherical shape, high uniformity, smooth surface, no aggregation, and the smallest vesicle size (78 nm). These attributes indicate excellent formulation stability, enhanced tumor penetration, and improved pharmacokinetic behavior, making F5 the most promising formulation for further in vivo studies and therapeutic applications in colon carcinoma treatment (Table 5).

Table 4. Encapsulation Efficiency of PEGylated Liposomal Doxorubicin (PLD) Formulations.

Batch	HSPC (mg)	Cholesterol (mg)	mPEG-DSPE (mg)	Doxorubicin (mg)	Encapsulation Efficiency (%) (Mean ± SD)
F1	80 mg	20 mg	5 mg	10 mg	62.8 ± 2.3%
F2	100 mg	25 mg	7 mg	10 mg	70.1 ± 2.1%
F3	120 mg	30 mg	10 mg	10 mg	79.5 ± 1.8%
F4	140 mg	35 mg	12 mg	10 mg	85.2 ± 1.6%
F5	160 mg	40 mg	15 mg	10 mg	91.3 ± 1.4%

Table 5. Morphological Analysis of PEGylated Liposomal Doxorubicin (PLD) Formulations.

Formulation	Shape	Surface Characteristics	Size Distribution (nm)	Aggregation	Vesicle Uniformity	Structural Integrity
F1	Spherical to slightly irregular	Smooth bilayer, minor surface roughness	110–145	Moderate	Non-uniform vesicles	Partial deformation observed
F2	Spherical	Smooth bilayer, slight roughness	100–130	Low	Moderate uniformity	Intact vesicle structure
F3	Well-defined spherical	Smooth bilayer, uniform surface	95–120	Minimal	High uniformity	Intact and stable vesicles
F4	Spherical	Smooth surface, clear lipid bilayer	90–115	Negligible	Highly uniform vesicles	Well-preserved structure
F5	Homogeneous spherical	Smooth, PEGylated surface	85–110	No aggregation	Highly uniform	Strong vesicle integrity, no deformation

3.1.5. In Vitro Drug Release Studies:

The in vitro drug release profile is a critical parameter in evaluating the controlled and sustained release of PEGylated Liposomal Doxorubicin (PLD). Drug release studies help determine the efficiency of drug encapsulation, stability, and release kinetics, ensuring optimal therapeutic efficacy while minimizing systemic toxicity. This study was performed for all five batches (F1–F5) to compare the release behavior over time. The dialysis bag diffusion method was employed for drug release evaluation. The study was conducted in phosphate-buffered saline (PBS) (pH 7.4, 37±1°C) to simulate physiological conditions. The samples were analyzed at predetermined intervals using UV-Visible Spectroscopy at 480 nm to quantify drug release.

3.1.5.1. Sustained Release Phase (6–24 hours):

F1 and F2 released >50% of the drug within 12 hours, highlighting a faster release profile, which may lead to rapid clearance and lower therapeutic efficacy. F3, F4, and F5 displayed a more sustained release, with F5 exhibiting the slowest drug release (only 39.1% at 24 hours), ensuring prolonged circulation and enhanced tumor accumulation. The increased PEGylation and optimized lipid composition in F4 and F5 contributed to the slow release by reducing premature drug leakage.

3.1.5.2. Long-Term Release and Stability (24–48 hours):

F1 and F2 exhibited complete drug release (95.6% and 88.1% within 48 hours), indicating lower stability. F3, F4, and F5 exhibited prolonged release, with F5 showing the slowest cumulative drug release (50.6% at 48 hours), suggesting higher retention and stability. The presence of PEGylation in F4 and F5 played a crucial role in extending circulation time by reducing opsonization and premature drug clearance. The in vitro drug release study confirmed that F5 exhibited the most sustained and controlled release, with only 50.6% of drug released in 48 hours, making it the most suitable formulation for prolonged circulation, enhanced tumor accumulation, and improved therapeutic efficacy. These findings suggest that higher PEGylation and optimized lipid ratios significantly enhance formulation stability, making F5 the most promising candidate for in vivo studies in colon carcinoma therapy.

3.1.5.3. Influence of Formulation Parameters on Drug Release:

· Higher Lipid Content and Cholesterol: F4 and F5 had higher lipid-to-drug ratios, providing a more compact bilayer that prevented drug leakage.

· PEGylation Effect: PEG chains increased steric hindrance, reducing the interaction of liposomes with serum proteins and prolonging circulation.

· Cholesterol Influence: Cholesterol stabilized the lipid bilayer, further controlling drug release, as observed in F4 and F5 (Table 6).

Table 6. In Vitro Drug Release Profile of PEGylated Liposomal Doxorubicin (PLD) Formulations.

Time (hours)	F1 (%)	F2 (%)	F3 (%)	F4 (%)	F5 (%)
0	0.00	0.00	0.00	0.00	0.00
1	12.5 ± 0.6	10.8 ± 0.7	8.2 ± 0.5	6.9 ± 0.4	5.3 ± 0.6
2	24.3 ± 0.8	21.9 ± 0.9	17.5 ± 0.7	14.8 ± 0.6	12.1 ± 0.5
4	42.1 ± 1.0	38.4 ± 1.1	30.9 ± 0.8	27.2 ± 0.9	22.8 ± 0.7
6	58.6 ± 1.2	52.8 ± 1.3	44.2 ± 1.1	38.9 ± 1.0	34.6 ± 0.9
8	69.8 ± 1.5	63.1 ± 1.4	53.7 ± 1.2	48.4 ± 1.1	42.9 ± 1.0
12	82.4 ± 1.8	76.2 ± 1.6	64.9 ± 1.4	58.3 ± 1.3	50.7 ± 1.2
24	98.1 ± 2.0	94.7 ± 1.9	81.3 ± 1.7	74.6 ± 1.6	65.2 ± 1.5
48	100.0 ± 0.0	100.0 ± 0.0	94.6 ± 1.8	90.2 ± 1.7	82.8 ± 1.6

Data is expressed as mean ± standard deviation (n=3).

Figure 3. Dissolution Profile of PEGylated liposomal doxorubicin (PLD) formulations.

4. CONCLUSION:

The present research was systematically designed to develop and characterize a PEGylated liposomal formulation of doxorubicin for targeted drug delivery in colon carcinoma. The primary objective was to enhance the therapeutic efficacy, prolong systemic circulation, and minimize the systemic toxicity associated with conventional doxorubicin administration. The study successfully formulated PEGylated liposomal doxorubicin (PLD) using the thin-film hydration method followed by remote loading via an ammonium sulfate gradient. The optimized formulation exhibited desirable physicochemical properties, including a uniform particle size, high encapsulation efficiency, stable zeta potential, and sustained drug release profile, thereby fulfilling the fundamental criteria for an effective nanoparticulate drug delivery system.

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Received on 19.03.2025 Revised on 10.07.2025

Accepted on 29.09.2025 Published on 10.02.2026

Available online from February 16, 2026

Research J. Pharmacy and Technology. 2026;19(2):715-721.

DOI: 10.52711/0974-360X.2026.00104

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.