Synthesis and characterisation of liposomal doxorubicin with loaded gold nanoparticles

Developing nanostructures for cancer treatment is growing significantly. Liposomal doxorubicin is a drug that is used in the clinic and represents a lot of benefits over doxorubicin. The development of multifunctional liposomes with different cancer treatment capability enables broader applications of doxorubicin chemotherapy. Many efforts were carried to prepare more effective liposomal formulation through loading gold nanoparticles (GNPs) in the formulation. Here, GNPs with an average size of 6 nm were loaded in liposomal formulation alongside doxorubicin. The hydrodynamic diameter of final formulation was 177.3 ± 33.9 nm that in comparison with liposomes without GNPs (112.5 ± 10.3 nm), GNPs‐loaded liposomes showed the bigger hydrodynamic diameter. GNPs‐loaded liposomes are slightly positively charged (4.4 ± 1.1 mV), while liposomes without loading the GNPs were negatively charged (−18.5 ± 1.6 mV). Doxorubicin was loaded in this formulation through active loading technique. Doxorubicin loading efficiency in gold‐loaded liposomes is slightly lesser than liposomes without GNPs, but still considerably high in comparison to passive loading techniques.Inspec keywords: nanofabrication, drugs, nanomedicine, gold, cancer, lipid bilayers, drug delivery systems, nanoparticles, crystal growth from solutionOther keywords: liposomal doxorubicin, multifunctional liposomes, doxorubicin chemotherapy, active loading technique, doxorubicin loading efficiency, gold‐loaded liposomes, passive loading techniques, gold nanoparticles, cancer treatment, liposomal formulation, GNP‐loaded liposomes, hydrodynamic diameter     

Preparation and characterization of lyophilised egg PC liposomes incorporating curcumin and evaluation of its activity against colorectal cancer cell lines

Curcumin has been associated with the treatment of various diseases in traditional medicine, among them cancer. The major problems that prevent its approval as therapeutic agent are its low water solubility and its relatively low in vivo bioavailability. Liposomes are considered as effective drug carriers because of their ability to solubilize hydrophobic compounds and to alter their pharmacokinetic properties. The purpose of this study was the development of lyophilised liposomal curcumin fully characterized in terms of its physical properties [(zeta-potential, size, size distribution and Polydispercity index (PI)], and to evaluate its in vitro cytotoxic against colorectal cancer cell lines in a short-term and in a long-term (clonogenic) assay. Curcumin was incorporated in egg-phosphatidylcholine (EPC) liposomes at a drug to lipid molar ratio 1:14 achieving high incorporation efficiency close to 85%. The liposomal curcumin was lyophilized preserving thus its stability. The reconstitution of the formulation resulted in the original liposomal suspension. The release in FBS showed a plateau near 14% at 96 hours of incubation. The in vitro studies against colorectal cancer cell lines have shown that liposomes improve the activity of curcumin especially in the long-term assay and the liposomal formulation found to be more potent against HCT116 and HCT15, cell lines which express the MDR phenotype. EPC liposomal curcumin in a molar ratio of curcumin/EPC 1:14 has shown improved cytotoxic activity versus free curcumin against colorectal cancer cell lines. In vivo studies based on the recent findings are in progress in our laboratory.     

Application of 99mTc Labeling for Fast Comparative Screening of Preformed Liposomes in vivo

Labeling of preformed liposomes with ^99mTc is a simple, quick, and highly versatile method, and its potential in the comparative screening of preformed liposomes with regard to their early biodistribution properties, such as the BLOOD/RES ratios at 2 and 30 min postadministration, was investigated. Liposomes differing in lipid composition and size were prepared and labeled under optimum conditions with ^99mTc using a slight modijication of a previously described method (1). The amount of SnCl₂ used to reduce pertechnetate affected liposome biodistribution. The labeling method employed was capable of detecting anticipated changes in liposome biodistribution caused by alterations in liposome composition or size. It could also reveal the effect of a relative immiscibility of monosialganglioside GM₁ with di-myristoy1-phosphatidyl-choline:di-myristoyl-phosphatidyl-glycerol (DMPC:DMPG) on the biodistribution of DMPC:DMPG:GM, liposomes. It is proposed that ^99mTc labeling of liposomes provides a tool for fast comparative screening of preformed liposomal preparations according to their early biodistribution.     

Application of 99mTc Labeling for Fast Comparative Screening of Preformed Liposomes in vivo

Abstract

Labeling of preformed liposomes with ^99mTc is a simple, quick, and highly versatile method, and its potential in the comparative screening of preformed liposomes with regard to their early biodistribution properties, such as the BLOOD/RES ratios at 2 and 30 min postadministration, was investigated. Liposomes differing in lipid composition and size were prepared and labeled under optimum conditions with ^99mTc using a slight modijication of a previously described method (1). The amount of SnCl₂ used to reduce pertechnetate affected liposome biodistribution. The labeling method employed was capable of detecting anticipated changes in liposome biodistribution caused by alterations in liposome composition or size. It could also reveal the effect of a relative immiscibility of monosialganglioside GM₁ with di-myristoy1-phosphatidyl-choline:di-myristoyl-phosphatidyl-glycerol (DMPC:DMPG) on the biodistribution of DMPC:DMPG:GM, liposomes. It is proposed that ^99mTc labeling of liposomes provides a tool for fast comparative screening of preformed liposomal preparations according to their early biodistribution.     

Preparation, characterization, and stability of liposome-based formulations of mitoxantrone

The preparation, characterization, and stability of lyophilized liposome-based formulation of mitoxantrone was investigated. Mitoxantrone was entrapped inside small, unilamellar liposomes composed of dioleoylphosphocholine (DOPC), cholesterol, and cardiolipin. The mean vesicle size and drug entrapment efficiency of the liposomes were ~ 150 nm and ~ 99%, respectively. Less than 1% of drug was lost and mean vesicle size remained unchanged after sterile filtration. The pre-lyophilized (pre-lyo) formulations were characterized by a differential scanning calorimetric (DSC) method. Results showed that the glass transition temperatures (Tg') increased as the molar ratios of sucrose:lipid and trehalose:lipid in the formulations were increased. The maximum Tg' of the pre-lyo formulations containing 10:1 sucrose:lipid and trehalose:lipid molar ratios were - 37°C and - 41°C, respectively. After reconstitution of the lyophilized cake of the sucrose-containing formulation, the mean vesicle size was comparable to pre-lyo liposome size. In vitro release studies showed that less than 2% of mitoxantrone was released after an extensive dialysis against phosphate buffered saline (PBS) at 37°C, indicating that the mitoxantrone was highly associated and retained inside the liposomes. Short-term stability studies of the sucrose-containing formulations revealed that the reconstituted and eight-fold diluted formulations were stable for up to 8 hours at room temperature. Long-term stability studies of lyophilized liposomal mitoxantrone showed that the lyophilized formulation was stable for up to 13 months after storage at refrigerated condition.     

Simultaneous Quantification of Tumor Uptake for Targeted and Nontargeted Liposomes and Their Encapsulated Contents by ICPMS

Liposomes are intensively being developed for biomedical applications including drug and gene delivery. However, targeted liposomal delivery in cancer treatment is a very complicated multistep process. Unfavorable liposome biodistribution upon intravenous administration and membrane destabilization in blood circulation could result in only a very small fraction of cargo reaching the tumors. It would therefore be desirable to develop new quantitative strategies to track liposomal delivery systems to improve the therapeutic index and decrease systemic toxicity. Here, we developed a simple and nonradiative method to quantify the tumor uptake of targeted and nontargeted control liposomes as well as their encapsulated contents simultaneously. Specifically, four different chelated lanthanide metals were encapsulated or surface-conjugated onto tumor-targeted and nontargeted liposomes, respectively. The two liposome formulations were then injected into tumor-bearing mice simultaneously, and their tumor delivery was determined quantitatively via inductively coupled plasma mass spectroscopy (ICPMS), allowing for direct comparisons. Tumor uptake of the liposomes themselves and their encapsulated contents was consistent with targeted and nontargeted liposome formulations that were injected individually.     

Liposomes containing drug and cyclodextrin prepared by the one-step spray-drying method

The one-step spray-drying method was applied in the preparation of liposomes containing drug and cyclodextrin (CD). Spray-dried lecithin liposomes, entrapping metronidazole or verapamil alone or together with hydroxypropyl-β-cyclodextrin (HPβCD), were characterized for morphology, size distribution, and drug entrapment efficiency. The main factor influencing the liposomal size was the volume of aqueous medium used for hydration of the spray-dried product. No differences in size or entrapment between liposomes prepared by immediate hydration of dried powder or by hydration after 1 year of powder storage at 4°C were observed. All liposomes were tested for their serum stability. The most stable liposomes (still retaining about 10% of the originally entrapped drug even after 24 hr incubation with serum) were liposomes prepared by the direct spray-drying of the mixture of lipid, drug, and HPβCD.     

Vesicular Lipidic Systems,Liposomes, PLO,and Liposomes–PLO: Characterization by Electronic Transmission Microscopy

Situations exist in which rapid administration of treatment, as well as maintenance of efficient concentrations for the longest possible time, turns out to be essential. In view of the previous treatment, the elaboration of liposomes, PLO (pluronic lecithin organogel), and the mixture of both is described, as well as their characterizations by electronic transmission microscopy, with the aim of finding out precisely the type of structure for both controlled release systems, its composition, size, homogeneity, and integrity. The period of study has been 90 days. Multilaminar and unilaminar vesicles smaller than 1 μm in diameter were seen in the liposomes, PLO, and liposomes–PLO formulations on transmission electron microscopic (TEM) observation. The technique of characterization reveals the progressive aggregation of the liposomas along the period of study. However, all the vesicles of PLO maintain a defined structure and only a light aggregation 60 days after the elaboration. Changes of morphology and aggregation of liposomas decreased after the incorporation of cholesterol (CH) to the liposomal matrix. The best results were obtained with the formulas liposomes–PLO, which maintain their individuality and integrity during the whole period of study. The combined formulation of liposomas and PLO showed an increase of stability of both lipid systems.     

Large-Scale Production of Liposomes of Defined size by a New Continuous High Pressure Extrusion Device

A new continuous high pressure extrusion apparatus for the generation of liposomes was designed and tested in the present study. The extruder, which basically consists of an open supply vessel and a high pressure filter holder mounted on a gas-driven pump exhibited superior abilities compared to currently available, discontinuous extrusion devices. Due to continuous flows up to 500 ml/min (filter diameter 47 mm), large batches on a liter scale could be extruded in one step. The maximum pressure of 10.5 MPa employed here, enabled the rapid passage of liposomal preparations with various lipid compositions at lipid concentrations as high as 400 mg/g through polycarbonate membranes without any clogging of the system. Employing final pore sizes between 5.0 and 0.03 μm, the mean vesicle diameter of liposomal preparations could be varied from 400 to 60 nm. Surprisingly high encapsulation efficiencies of two water-soluble contrast agents, iopromide and Gd-DTPA, which we used as model substances, were obtained for continuously extruded liposomes. Values of over 50 % for iopromide and more than 60 % for Gd-DTPA, which were achieved in combination with a freeze-thaw protocol, to our knowledge exceed all results reported so far for passive entrapment of water-soluble contrast agents. The generation of 6 batches of iopromide-carrying liposomes under identical conditions revealed good reproducibility of the new method. Physico-chemical characterization of void as well as contrast-carrying liposomes after storage at 2–8°C for 6 months showed satisfactory long-term stability of continuously extruded liposomes.

The new structural design of the high pressure extrusion device permits, for the first time, reproducible, continuous, large-scale production of stable liposomes of defined size. Scaling up the widely used extrusion method to an industrial level seems feasible now.     

Large-Scale Production of Liposomes of Defined size by a New Continuous High Pressure Extrusion Device

A new continuous high pressure extrusion apparatus for the generation of liposomes was designed and tested in the present study. The extruder, which basically consists of an open supply vessel and a high pressure filter holder mounted on a gas-driven pump exhibited superior abilities compared to currently available, discontinuous extrusion devices. Due to continuous flows up to 500 ml/min (filter diameter 47 mm), large batches on a liter scale could be extruded in one step. The maximum pressure of 10.5 MPa employed here, enabled the rapid passage of liposomal preparations with various lipid compositions at lipid concentrations as high as 400 mg/g through polycarbonate membranes without any clogging of the system. Employing final pore sizes between 5.0 and 0.03 μm, the mean vesicle diameter of liposomal preparations could be varied from 400 to 60 nm. Surprisingly high encapsulation efficiencies of two water-soluble contrast agents, iopromide and Gd-DTPA, which we used as model substances, were obtained for continuously extruded liposomes. Values of over 50 % for iopromide and more than 60 % for Gd-DTPA, which were achieved in combination with a freeze-thaw protocol, to our knowledge exceed all results reported so far for passive entrapment of water-soluble contrast agents. The generation of 6 batches of iopromide-carrying liposomes under identical conditions revealed good reproducibility of the new method. Physico-chemical characterization of void as well as contrast-carrying liposomes after storage at 2-8°C for 6 months showed satisfactory long-term stability of continuously extruded liposomes.

The new structural design of the high pressure extrusion device permits, for the first time, reproducible, continuous, large-scale production of stable liposomes of defined size. Scaling up the widely used extrusion method to an industrial level seems feasible now.     

Liposomes Containing Drug and Cyclodextrin Prepared by the One-Step Spray-Drying Method

The one-step spray-drying method was applied in the preparation of liposomes containing drug and cyclodextrin (CD). Spray-dried lecithin liposomes, entrapping metronidazole or verapamil alone or together with hydroxypropyl-β-cyclodextrin (HPβCD), were characterized for morphology, size distribution, and drug entrapment efficiency. The main factor influencing the liposomal size was the volume of aqueous medium used for hydration of the spray-dried product. No differences in size or entrapment between liposomes prepared by immediate hydration of dried powder or by hydration after 1 year of powder storage at 4°C were observed. All liposomes were tested for their serum stability. The most stable liposomes (still retaining about 10% of the originally entrapped drug even after 24 hr incubation with serum) were liposomes prepared by the direct spray-drying of the mixture of lipid, drug, and HPβCD.     

Development of a Liposome Based Contraceptive System for Intravaginal Administration of Progesterone

Progesterone bearing liposomes were prepared by the cast film method and characterized for various physical attributes. The liposomes could encapsulate nearly 98% of the drug. The progesterone bearing liposomes were incorporated into polyacrylamide gel and the drug content was determined. The in vitro drug dqfusion across she-goat vaginal membrane from the liposomal formulation was found to follow near zero order kinetics. The progestational activity of liposomal and control gel was assessed by monitoring the effect on the formation of corpora lutea. It was observed that both the formulations inhibit the formation of corpora lutea and thus, exhibited progestationul activity but the efsect of liposomal preparation was found to be greater and prolonged as compare to control gel.     

Effect of PEGylation on the stability of liposomes during nebulisation and in lung surfactant

Oral inhalation of anticancer drugs or drug delivery system is a novel therapeutic approach in the treatment of lung cancer and requires formulations which are sufficiently stabile during nebulisation and subsequent interaction with the surfactant lining of the lungs. In this study, we assessed the stability of plain and PEGylated transferrin-conjugated liposomes after nebulisation using two different nebulisers (i.e., air-jet and ultrasonic type). Furthermore, the integrity of the liposomal membranes was assessed after incubation in commercial lung surfactant solutions (Alveofact). All liposomal formulations showed no significant changes in their size after nebulisation, independent of the type of nebuliser or the liposomal formulation, respectively. However, PEGylation was of advantage when it came to interactions between liposomes and the surfactant lining of the lungs. PEGylated liposomes were significantly more stable and retained > 80% of their drug load over 48 h, which is more than sufficient time for the drug carriers to be taken up by transferrin receptor over-expressing cancer cells in the lung. In conclusion, PEGylated and plain Tf-conjugated liposomes are stable enough to undergo nebulisation in the course of an inhalational therapy, but PEG-stabilisation results in a higher degree of membrane integrity in lung surfactant.     

Preparation,Characterization, and Evaluation of Liposomal Dispersions of Lidocaine

Abstract

Lidocaine, a local anesthetic agent, was encapsulated into liposomes employing the conventional lipid-film hydration technique. An attempt was made to freeze dry the aqueous liposomal dispersions. The prepared liposomal dispersions were investigated by differential scanning calorimetry (DSC), transmission electron microscopy (TEM), scanning electron microscopy (SEM), ³¹ P-nuclear magnetic resonance (NMR) spectroscopy, and laser counting studies for characterization. The skin partition coefficient for liposomal lidocaine was calculated. The results showed that lidocaine incorporated into the liposomes got selectively partitioned and localized in the skin to a great extent. A topical liposomal gel formulation containing 2% w/w lidocaine was prepared using Carbopol-934 as the gelling agent. The prepared formulation was tested for its local anesthetic efficacy employing the pinprick test. The liposomal preparation of lidocaine gave a much longer duration of action compared to the conventional topical formulation.     

Vesicular lipidic systems, liposomes, PLO, and liposomes-PLO: characterization by electronic transmission microscopy

Situations exist in which rapid administration of treatment, as well as maintenance of efficient concentrations for the longest possible time, turns out to be essential. In view of the previous treatment, the elaboration of liposomes, PLO (pluronic lecithin organogel), and the mixture of both is described, as well as their characterizations by electronic transmission microscopy, with the aim of finding out precisely the type of structure for both controlled release systems, its composition, size, homogeneity, and integrity. The period of study has been 90 days. Multilaminar and unilaminar vesicles smaller than 1 microm in diameter were seen in the liposomes, PLO, and liposomes-PLO formulations on transmission electron microscopic (TEM) observation. The technique of characterization reveals the progressive aggregation of the liposomas along the period of study. However, all the vesicles of PLO maintain a defined structure and only a light aggregation 60 days after the elaboration. Changes of morphology and aggregation of liposomas decreased after the incorporation of cholesterol (CH) to the liposomal matrix. The best results were obtained with the formulas liposomes-PLO, which maintain their individuality and integrity during the whole period of study. The combined formulation of liposomas and PLO showed an increase of stability of both lipid systems.     

Preparation, Characterization, and Evaluation of Liposomal Dispersions of Lidocaine

Lidocaine, a local anesthetic agent, was encapsulated into liposomes employing the conventional lipid-film hydration technique. An attempt was made to freeze dry the aqueous liposomal dispersions. The prepared liposomal dispersions were investigated by differential scanning calorimetry (DSC), transmission electron microscopy (TEM), scanning electron microscopy (SEM), ³¹ P-nuclear magnetic resonance (NMR) spectroscopy, and laser counting studies for characterization. The skin partition coefficient for liposomal lidocaine was calculated. The results showed that lidocaine incorporated into the liposomes got selectively partitioned and localized in the skin to a great extent. A topical liposomal gel formulation containing 2% w/w lidocaine was prepared using Carbopol-934 as the gelling agent. The prepared formulation was tested for its local anesthetic efficacy employing the pinprick test. The liposomal preparation of lidocaine gave a much longer duration of action compared to the conventional topical formulation.     

Preparation, characterization and in vivo studies of proliposomes containing Cyclosporine A

The present study was undertaken to prepare proliposomes of Cyclosporine A (CsA) to increase its oral bioavailability. The proliposomes were prepared by spraying a solution of CsA, egg lecithin and cremophor EL in methanol-chloroform mixture onto directly compressible lactose (carrier) in a rotary evaporator. A dry free flowing powder of proliposomes was obtained. The dry proliposomal powder was characterized for surface morphology by scanning electron microscopy (SEM). Then the proliposomes were hydrated with distilled water to produce liposomes, which were characterized for particle size distribution, % drug entrapment, and morphological characteristics by transmission electron microscopy (TEM). The liposomes exhibited good entrapment of about 99%. The entrapment of CsA in liposomes was found to be dependent mainly on the drug:lipid ratio. Bioavailability studies were carried out for three different formulations of CsA i.e., free drug suspension; proliposomes derived liposomes and marketed formulation (Pannimun Bioral, Microemulsion) on male SD rats. The results of bioavailability studies indicated that the difference in the mean drug concentration of the free drug and the liposomes was found to be statistically significant (p < 0.05, p value is 0.032). The absorption constant for liposomal product was much greater (10.26 h(-1)) than for free drug solution (1.2 h(-1)) or the marketed sample of microemulsion (2.51 h(-1)) and the volume of distribution was found to be less for liposomes (7629.88 ml/kg) than that of the free drug solution (10971.92 ml/kg) and marketed microemulsion (9012.07 ml/kg). The results of these studies have shown that a stable proliposomal formulation of CsA for oral administration can be prepared which can be easily hydrated into liposomes from which CsA can exert its clinical effects with a better oral bioavailability.     

Preparation and pharmaceutical/pharmacodynamic evaluation of topical brucine-loaded liposomal hydrogel

To reduce the toxicity and enhance the therapeutic efficacy of brucine, a traditional Chinese medicine for relieving arthritic and traumatic pain, in this study, a novel brucine-loaded liposomal hydrogel (BLH) formulation, suitable for topical application, was developed. Spherical liposomes composed of lecithin and cholesterol, with brucine, was prepared by a modified ethanol-dripping method. High percentage (over 80%) of encapsulated brucine in liposomes was obtained. Topical liposomal hydrogel formulations were prepared by further incorporation of the prepared liposomes into structured carbopol 940 hydrogels with the concentration of carbopol 1.0%, the ratio of glycerol to carbopol 8:1 and the brucine content 0.1%. The liposomal hydrogel formulations provided an obvious promotion for skin permeation of bruicne while for the free brucine in hydrogels (BH), there was no detectable drug permeation through the skin. The safety evaluation showed that the prepared BLH were no irritation to both the broken and integrity skin. Pharmacodynamic evaluation revealed that the BLH showed a better therapeutic efficacy than that of the BH. So, it can be concluded that the BLH developed here could represent a safe, effective and promising transdermal formulation for local treatment of analgesic and anti-inflammatory disease.     

Continuous release of interleukin-2 from liposomal IL-2 (mixture of interleukin-2 and liposomes) after subcutaneous administration to mice

Recombinant interleukin-2 (IL-2) was strongly and almost completely adsorbed onto small and hydrophobic liposomes by simple mixing under optimal conditions (liposome: DSPC-DSPG; molar ratio, 10:1; 30-50 nm in size, ratio of IL-2 to liposome: 4.0 JRU/nmol lipid). This liposomal IL-2 displayed better distribution after intravenous administration in mice and improved therapeutic effect against experimental M5076 metastases, as reported previously.[1] In this study, the elimination of IL-2 from the dosing area was investigated when the liposomal IL-2 was administered to mice subcutaneously. The results suggest that the release of IL-2 from this liposome was continuous and almost complete. The mean residence time (MRT) of IL-2 in the dosing area was 11.0 ± 1.65 hr. This resulted in the 8-fold times enhancement of MRT in the systemic circulation by the presence of liposomes, and IL-2 was detected in the serum for 2 days. Using this liposomal IL-2 is expected to have the potential to decrease the number of injections and enhance the efficacy of IL-2 in immunotherapies and therapies against tumor.     

Intradermal and follicular delivery of adapalene liposomes

Abstract

Background: Adapalene is a widely used topical anti-acne drug; however, it has many side effects. Liposomal drug delivery can play a major role by targeting delivery to pilosebaceous units, reducing side effects and offering better patient compliance.

Objective: To prepare and evaluate adapalene-encapsulated liposomes for their physiochemical and skin permeation properties.

Methods: A liposomal formulation of adapalene was prepared by the film hydration method and characterized for shape, size, polydispersity index (PDI), encapsulation efficiency and thermal behavior by techniques such as Zetasizer®, differential scanning calorimetry and transmission electron microscopy. Stability of the liposomes was evaluated for three months at different storage conditions. In vitro skin permeation studies and confocal laser microscopy were performed to evaluate adapalene permeation in pig ear skin and hair follicles.

Results: The optimized process and formulation parameters resulted in homogeneous population of liposomes with a diameter of 86.66?±?3.5?nm in diameter and encapsulation efficiency of 97.01?±?1.84% w/w. In vitro permeation studies indicated liposomal formulation delivered more drug (6.72?±?0.83?μg/cm²) in hair follicles than gel (3.33?±?0.26?μg/cm²) and drug solution (1.62?±?0.054?μg/cm²). Drug concentration delivered to the skin layers was also enhanced compared to other two formulations. Confocal microscopy images confirmed drug penetration in the hair follicles when delivered using the liposomal formulation.

Conclusion: Adapalene was efficiently encapsulated in liposomes and led to enhanced delivery in hair follicles, the desired target site for acne.