Current Nanomaterials

Current Nanomaterials

62

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Current Nanomaterials 2018, 3, 62-74

REVIEW ARTICLE ISSN: 2405-4615 eISSN: 2405-4623

Recent Developments in the Formulation of Nanoliposomal Delivery Systems BENTHAM SCIENCE

Mandeep Dahiya and Harish Dureja* Department of Pharmaceutical Sciences, Maharshi Dayanand University, Rohtak - 124001, India

ARTICLE HISTORY Received: July 24, 2018 Revised: August 10, 2018 Accepted: August 13, 2018 DOI: 10.2174/2405461503666180821093033

Abstract: Nanoliposomes, or submicron bilayer phospholipid vesicles, are a novel tool to encapsulate and deliver the active pharmaceutical ingredients. The listing of active substances for integration into nanoliposomes is enormous, varying from pharmaceuticals to nutraceuticals and cosmetics. Due to their biocompatible and biodegradable nature, besides their nanosize, nanoliposomes have applications in diverse areas, like cancer nanotherapy, cosmetics, gene delivery and agricultural food technology. Nanoliposomes have the capability of improving the efficacy of active agents by recuperating there in vitro-in vivo solubility, bioavailability, stability and prevent unnecessary interactions between molecules. Targeting by nanoliposomes depends on the types of the cells to achieve active drug concentrations for favorable therapeutic effectiveness at the desired target site and minimizing undesirable effects on healthy tissues and cells. This article sums up the existing studies that have described the nanoliposomal particles as carriers to transport a variety of pharmaceutical agents, properties, methods of preparation, and analysis.

Keywords: Docetaxel, doxorubicin, gene delivery, magnetic, nanoliposome, PEG, tumor. 1. INTRODUCTION

1.1. Advantages of Nanoliposomes Based Approaches [5]:

Among the drug-delivery systems available, nanoliposomes (artificial phospholipids vesicles) comprise a bilayered membrane configuration (Fig. 1) that have got an immense concern as superior and flexible pharmaceutical nanocarriers for low and high molecular weight active agents [1]. They are produced by different methods like dispersions of lipids in water and able to encapsulate the active pharmaceutical drug. Nanoliposomes are preferred as carriers of the active pharmaceutical drug having plenty of functional clinical applications [2].

1. Liposomes increased efficacy and therapeutic index of drug (e.g., actinomycin-D).

Nanoliposomes are biologically compatible, cause slight or no pyrogenic, antigenic, toxic and hypersensitivity reactions. Nanoliposomes undergo biodegradation and shield the patient from the adverse effects of the encapsulated medicinal agent and protect the entrapped active agent from the neutralizing action of the physiological medium. Nanoliposomes are competent in delivering the active content within the cells [3]. Along with lipids and phospholipid molecules, sterols may be present in the nanoliposome structure, which are the essential constituent of the cell membrane. Cholesterol is the most widely used sterol, which can be integrated into phospholipid membranes in exceptionally higher concentrations, for example, up to 1:1 molar ratios of phospholipid to cholesterol [4].

*Address correspondence to this author at the Department of Pharmaceutical Sciences, Maharshi Dayanand University, Rohtak, Haryana, India; Tel: +91 9416357995; E-mail: [email protected]

2405-4623/18 $58.00+.00

2. Flexibility to couple with site-specific ligands to achieve active targeting. 3. Liposome increased stability via encapsulation. 4. Liposomes are non-toxic, flexible, biocompatible, biodegradable, and non-immunogenic for systemic and nonsystemic administrations. 5. Liposomes reduce the toxicity of the encapsulated agent (amphotericin B, taxol). 6. Liposomes help reduce the exposure of sensitive tissues to toxic drugs. 7. Site avoidance effect. Biodistribution factor of nanoliposomes is a significant factor from the clinical aspect. Nanoliposomes can vary the distribution in tissues, and the rate of drug clearance [6]. The pharmacokinetic profile of the nanoliposomes is reliant on their physicochemical characteristics, like surface charge, particle size, packing of lipid, stability, dose, and its administration routes. Conventional nanoliposomes are susceptible to removal from the systemic circulation via the Reticulo Endothelial System (RES) [7]. After the intravenous administration of nanoliposomes, a major dose portion is absorbed through the Kupffer cells of the Reticulo Endothelial System (RES) found in the liver [8]. Conventional liposomes accumulate principally in a tumor, liver, and spleen [9]. Clini© 2018 Bentham Science Publishers

Nanoliposomal Delivery Systems

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Targeting peptide/molecule PEG coating Lipid membrane Internal aqueous space Drug payload

Fig. (1). A basic structure of nanoliposome.

cally approved and clinical trials liposomal formulations are shown in (Tables 1 and 2), respectively. To accumulate the nanoliposomes at the required sites, surface-attached ligands competent to distinguish and binds to the desired cells have been used. Targeted liposomes present many benefits over individually targeted drugs by using polymers [47]. The most convincing advantage is the increased drug loading. The combination of various ligand molecules on the surface of nanoliposome can be modified to improve the drug uptake. IgG class immunoglobulins are the extensively used components in the drug targeting. They offer a “bystander kill” outcome due to the diffusion of the active medicinal agent into adjacent tumor cells. They don't affect the liposome integrity [48]. In the present manuscript, various formulation aspects and applications of nanoliposomes as delivery systems are described. 2. SURFACE MODIFIED NANOLIPOSOMES FOR VARIOUS DISORDERS To provide a long circulation to nanoliposomes, various techniques have been used, for e.g., the nanoliposome surface is coated with inactive, biocompatible polymers, like Polyethylene Glycol (PEG), to provide a defensive coating on the surface and decelerate the liposome detection by opsonins and consequential clearance. The polyethylene glycol chains on the nanoliposomal surface prevent the vesicle agglomeration; hence, provides an improvement in the stability of formulations [49]. PEGylated nanoliposomes exhibit kinetics, which is linear, dose-independent and non-saturable with improved bioavailability. PEG is regarded as the best polymer to provide steric protection to nanoliposomes. Subcutaneous application of PEGylated nanoliposomes has the potential to target the lymph nodes, attaining continuous drug release in vivo [50]. PEGylated nanoliposomes of mupirocin avoids its speedy hydrolysis in vivo, expands its use in the parenteral formulation and targets it by the improved

permeability result to the affected tissue. Propylene glycol and hydroxypropyl-β-cyclodextrin were found to increased mupirocin loading [51]. Nanoliposomes of artemisinin were prepared by reverse phase evaporation were PEGylated by using polyethylene glycol 2000 in order to increase its stability and solubility. It was found that the cytotoxicity effect of PEGylated nanoliposomal artemisinin was more in breast cancer cells, in comparison with nanoliposomal artemisinin [52]. Resveratrol along with 5-fluorouracil was coencapsulated in a distinct nanoliposome containing PEG to check their synergistic therapeutic potential. The in vitro analysis on NT8e (cell line for the neck and head cancer) was found to demonstrate a GI50 alike that of free 5fluorouracil. The mixture of 5-fluorouracil and resveratrol showed diverse distinctive effects on many genes responsible for affecting the overall antagonistic outcome. In vitro testing showed that encapsulating both the drugs in nanoliposomal form improved the cytotoxicity in contrast to the free active drug mixture [53]. PEGylated nanoliposomes with angiogenic peptides can target an ischemic myocardium. Surface modifications with PEG increased myocardial uptake of ~100 nm nanoliposomes and improved myocardial perfusion defects and augmented vascular density [54]. To develop a sterically stable, targeting proteoliposome, a postinsertion method was employed to biofunctionalize the liposome surface with a Biocompatible Anchor Molecule (BAM) linker conjugated with Epidermal Growth Factor (EGF) as a homing molecule. Proteoliposomes biofunctionalized with the BAM-EGF complex encapsulating Cy5 fluorescent dye were selectively bound to the surface of MDAMB-231 cells overexpressing EGFR (Epidermal Growth Factor Receptor), but not to MCF-7 cells, which did not express EGFR. The same proteoliposome encapsulating doxorubicin was selectively targeted to MDAMB-231 cells and killed them. BAM could be used as a suitable postinsertion linker for biofunctionalization of liposome surface with high modification efficiency and stability [55].

64Current Nanomaterials, 2018, Vol. 3, No. 2

Table 1.

Dahiya and Dureja

Liposomal drugs approved for clinical applications.

Active Drug/Product Name

Lipid Composition

Indications

References

Daunorubicin (DaunoXome)

DSPC and Cholesterol (2:1 molar ratio)

AIDS-related Kaposi’s sarcoma

[10]

Doxorubicin (Doxil, Caelyx)

HSPC:Cholesterol:PEG 2000-DSPE (56:39:5 molar ratio)

Refractory Kaposi’s sarcoma; ovarian cancer and recurrent breast

[11]

Cytarabine/Ara-C (Depocyt)

DOPC, DPPG, Cholesterol, and Triolein

Neoplastic meningitis

[12]

Doxorubicin (Myocet)

EPC:Cholesterol (55:45 molar ratio)

Combination therapy with cyclophosphamide in metastatic breast cancer

[13]

Doxorubicin (Lipo-dox)

DSPC, cholesterol, and DSPE-PEG2000

Kaposi’s sarcoma, ovarian and breast cancer

[14]

Mifamurtide (Mepact)

DOPS: POPC (3:7 molar ratio)

High-grade, resectable, non-metastatic osteosarcoma

[15]

Vincristine (Marqibo)

SM: Cholesterol (60:40 molar ratio)

Metastatic malignant uveal melanoma, Acute lymphoblastic leukemia

[16, 17]

Amphotericin B (Abelcet)

DMPC: DMPG (7:3 molar ratio)

Invasive severe fungal infections

[18]

Amphotericin B (Amphocil)

An equimolar mixture of amphotericin B and cholesteryl sulfate

Fungal infections

[19]

Amphotericin B (Amphotec)

Cholesteryl sulphate:Amphotericin B (1:1 molar ratio)

Severe fungal infections

[20]

Verteporphin (Visudyne)

Verteporphin:DMPC and EPG (1:8 molar ratio)

Choroidal neovascularisation

[21]

Morphine sulphate (DepoDur)

DOPC, DPPG, Cholesterol and Triolein

Pain management

[22]

Bupivacaine (Exparel)

DEPC, DPPG, Cholesterol and Tricaprylin

Pain management

[23]

Irinotecan (Onivyde)

DSPC:MPEG-2000:DSPE (3:2:0.015 molar ratio)

Combination therapy withfluorouracil and leucovorin in metastatic adenocarcinoma of the pancreas

[24]

Amphotericin B (AmBisome)

HSPC:DSPG:Cholesterol:Amphotericin B (2:0.8:1:0.4 molar ratio)

Presumed fungal infections

[25]

Inactivated hepatitis A virus (strain RGSB) (Epaxal®)

DOPC:DOPE (75:25 molar ratio)

Hepatitis A

[26]

Inactivated hemaglutinine of Influenza virus strains A and B (Inflexal®)

DOPC:DOPE (75:25 molar ratio)

Influenza

[27]

Abbreviations: DSPC (distearoylphosphatidylcholine); HSPC (hydrogenated soy phosphatidylcholine); PEG (polyethylene glycol); DSPE (distearoyl-sn-glycero phosphoethanolamine); DOPC (dioleoylphosphatidylcholine); DPPG (dipalmitoylphosphatidylglycerol); EPC (egg phosphatidylcholine); DOPS (dioleoylphosphatidylserine); POPC (palmitoyloleoylphosphatidylcholine); SM (sphingomyelin); DMPC (dimyristoyl phosphatidylcholine); DMPG (dimyristoyl phosphatidylglycerol); DEPC (dierucoylphosphatidylcholine); MPEG (methoxy polyethylene glycol); DOPE (dioleoly-sn-glycero-phophoethanolamine); DPPC (Dipalmitoyl phosphatidylcholine); DOTAP (dioleoyloxypropyltrimethylammonium).

2.1. Magnetic Nanoliposomes Magnetoliposomes (MLs) are made up of nanosized, magnetizable iron oxide cores, which are distinctively encircled by a double layer of phospholipid molecules [56]. Intravenous administration of magnetic doxorubicin nanoliposomes was used to treat osteosarcoma-bearing hamsters. A limb with an implanted tumor, showed a fourfold rise in the drug concentration when positioned between the poles of the magnet (0.4 Tesla) for 60 min [57]. In a magnetofection process, self-aggregating conglomerates of cationic lipids with plasmid DNA or small interfering RNA (siRNA) was coupled with magnetic nanoparticles and concentrated

on the plane of cultured cells by the application of an undeviating inconsistent magnetic field. The procedure resulted in a significant enhancement in transfection efficiency compared to transfection performed with non-magnetic gene vectors [58]. Artemisinin in combination with transferrin exhibits anti-cancer activities. Magnetic nanoliposomes were prepared by an extrusion method, in thermosensitive and non-thermosensitive forms and evaluated for their antiproliferative activity against MCF-7 and MDA-MB-231 cells for improved tumor-targeted treatment. The thermosensitive formulation showed an appropriate state for thermal drug release at 42°C and demonstrated elevated antiproliferative activity against MCF-7 and MDA-MB-231 cells under the


Table 2.


65

Liposomal formulations present in clinical trials.

Active Drug/Product Name/Phase

Lipid Composition

Indications

References

Amikacin (Arikace) (Phase III)

DPPC and cholesterol

Lung infections

[28]

Tecemotide (Stimuvax) (Phase III)

Cholesterol, DMPG, DPPC

Non-small cell lung cancer

[29]

T4 endonuclease V (T4N5 liposomal Lotion) (Phase III)

Egg lecithin

Xeroderma pigmentosum

[30]

Doxorubicin (ThermoDox) (Phase III)

DPPC, Myristoyl stearyl phosphatidylcholine and DSPE-N-[amino(polyethylene glycol)-2000]

Hepatocellular carcinoma and also recurring chest wall breast cancer

[31]

Cisplatin (Lipoplatin) (Phase III)

DPPG, soy phosphatidylcholine, mPEG-distearoyl phosphatidylethanolamine lipid conjugate, and cholesterol

Non-small cell lung cancer

[32]

Platinum analogue cis-(trans-R,R-1,2diaminocyclohexane) bis (neodecanoato) platinum (II) (Aroplatin), (Phase II)

DMPC and DMPG

Metastatic colorectal cancer

[33]

Cisplatin (SPI-077), (Phase II)

Soybean phosphatidylcholine, cholesterol

Lung, head and neck cancer

[34]

Semi-synthetic doxorubicin analogue annamycin, (Annamycin), (Phase II)

DMPC and DMPG

Relapsed or refractory acute myeloid leukemia

[35]

Potent topoisomerase I inhibitor, (SCKD602), (Phase II)

Phospholipids covalently bound to mPEG

Cancer

[36]

Lurtotecan, (OSI-211), (Phase II)

HSPC and cholesterol

Ovarian, head and neck cancer

[37]

Irinotecan’s active metabolite, (LE-SN38), (Phase II)

DOPC, cholesterol, and cardiolipin

Advanced colorectal cancer

[38]

Paclitaxel, (Endotag-I), (Phase II)

DOTAP: DOPC: Paclitaxel

Breast and pancreatic cancers

[39]

Paclitaxel, (LEP-ETU), (Phase II)


Cancer

[40]

All-trans retinoic acid (Atragen), (Phase II)

DMPC and soybean oil

Hormone-resistant prostate cancer, renal cell carcinoma, and acute myelogenous leukemia

[41]

Mitoxantrone, (LEM-ETU), (Phase I)


Various Cancers

[42]

Vinorelbine, (Alocrest), (Phase I)

Sphingomyelin/cholesterol (OPTISOME™)

Breast and lung cancers

[43]

Vinorelbine tartrate, (INX-0125), (Phase I)

Cholesterol and sphingomyelin

Advanced solid tumors

[44]

p53 gene, (SGT-53), (Phase I)

Cationic lipids complexed with plasmid DNA encoding wild-type p53 tumor suppressor protein

Various solid tumors

[43]

Topotecan, (INX-0076), (Phase I)

Cholesterol and sphingomyelin


[44]

PLK1 siRNA, (TKM-080301), (Phase I)

Unique LNP technology (formerly referred to as stable nucleic acid-lipid particles or SNALP)

Neuroendocrine tumors

[45]

Doxorubicin, (2B3-101), (Phase I)

Glutathione PEGylated liposomes

Solid tumors

[46]

Docetaxel, (ATI-1123), (Phase I)

Protein stabilizing liposomes (PSL™)

Solid tumors

[43]

CEBPA siRNA, (MTL-CEBPA), (Phase I)

SMARTICLES® liposomal nanoparticles

Liver cancer

[43]

Doxorubicin, (MCC-465), (Phase I)

DPPC, cholesterol and maleimidated palmitoyl phosphatidyl ethanolamine; immunoliposomes tagged with PEG and the F(ab0)2 fragment of human monoclonal antibody GAH

Metastatic stomach cancer

[43]

Cisplatin, (LiPlaCis), (Phase I)

The lipid composition of the LiPlasomes is tailored to be specifically sensitive to degradation by the sPLA2 enzyme


[43]


effect of the magnetic field [59]. The combination of transferrin and artemisinin has anticancer properties. In vivo, antibreast cancer action of artemisinin and transferring loaded magnetic nanoliposome against breast relocated tumors in BALB/c mice model was investigated. Segments of tumor tissue were transplanted subcutaneously. Two hours after this treatment, artemisinin and transferrin loaded magnetic nanoliposomes were administered intravenously. Compared with free artemisinin and transferrin, the formulation could decrease the tumor volume in mice after treatment [60]. For Magnetic Resonance Imaging (MRI) and gammascintigraphy, an adequate amount of a radionuclide coupled with the nanoliposome, to attain an elevated signal to noise ratio is required. To get a high efficiency of nanoliposomes for contrast medium, two routes can be used. First, to raise the amount of carrier metal (111In or Gd), or increase the strength of the signal. Chelating amphiphilic polymers, for e.g., N, e-(DTPA-poly lysyl) glutaryl phosphatidylethanolamine can be used to enhance the nanoliposomal loading with reporter metals [61]. 99mTc has a short half-life with perfect radioactivity; it is, therefore, clinically recommended isotope for gamma-scintigraphy. Highly effective and stable glutathione liposomes with 99mTc and 186Re complexes were formulated [62]. Nanoliposomes for sonography are produced via integrating gas bubbles into the liposome. Gas bubbles can be formed directly inside the liposomes by bicarbonate hydrolysis producing carbon dioxide. In rabbit and porcine models, superior performance and lower toxicity of the contrast agents in the form of bubbles stabilized within the phospholipid membrane were achieved [63]. Hydrophilic doxycycline HCl (DOX) and hydrophobic drug raloxifene HCl (RAL), two potentially synergistic agents in the treatment of osteoporosis and other bone lesions, in combination with a radio frequency-induced, a hydrophobic magnetic nanoparticle-dependent triggering mechanism for drug release, were formulated. Drugs were incorporated into liposomes by lipid film hydration. Liposome stability was improved by reducing the drug load and by including Pluronics® (PL) in the formulations. DOX did not appear to interact with the phospholipid membranes comprising the liposomes, and its release was maximized in the presence of Radio Frequency (RF) heating. In contrast, Differential Scanning Calorimetry (DSC) and Phosphorus-31 Nuclear Magnetic Resonance (31P-NMR) analysis revealed that RAL developed strong interactions with the phospholipid membranes. Likewise, RAL release from liposomes was minimal, even in the presence of RF heating [64]. The 188Re-labeled pegylated nanoliposomes were assessed as a theranostic agent for 188 Re-N, N-bis(2-mercaptoethyl)-N′, N′glioma. diethylethylenediamine complex was laden on the pegylated liposome core with pH 5.5 ammonium sulfate gradient to produce 188Re-Liposome. Orthotopic Fischer344/F98 glioma tumor-bearing rats were injected intravenously with 188ReLiposome. Computed tomography gives a clear tumor image of the liposomal accumulation within the brain and proves the potential of 188Re-Liposome as a theranostic agent for the brain glioma [65]. The novel magnetic nanosized liposomes comprising of the PEI-As2O3/Mn0.5Zn0.5Fe2O4 complexes were prepared and characterized. As2O3/Mn0.5Zn0.5Fe2O4 and Mn0.5Zn0.5 Fe2O4 nanoparticles were synthesized by chemical coprecipitation

Dahiya and Dureja

method and loaded with PEI. PEI-As2O3/Mn0.5Zn0.5Fe2O4 were an applicable carrier for the delivery of a foreign gene to HepG2 cells. Upon experience to an alternating magnetic field, the Mn0.5Zn0.5Fe2O4 nanoparticles show good magnetic responsiveness, even after being modified by PEI and enclosed in liposomes. Results show that PEI-As2O3/ Mn0.5Zn0.5Fe2O4 magnetic nanoliposomes are brilliant biomaterial with various benefits in gene therapy, chemotherapy and tumor thermotherapy [66]. The transferrin and artemisinin-loaded magnetic nanoliposomes were prepared and evaluated for their antiproliferative activity against MCF-7 and MDA-MB-231 cell lines in vitro. The prepared nanoliposomes can significantly decrease the tumor volume in mice that are tumorized at 15 days after treatment as well as induce apoptosis in the mice breast cancer cells close to an external magnetic field. This information says that the transferrin and artemisinin-loaded magnetic nanoliposomes are an excellent option for the breast tumor-targeted therapy because of its great targeting efficiency [67]. 2.2. pH-sensitive Nanoliposomes pH-sensitive nanoliposomes are commonly used to deliver the drugs inside the cells. After being endocytosed in the undamaged shape, liposome integrates with the endovacuolar membrane due to lowered pH within the endosome and releases the loaded drug into the cytoplasm [33]. Pathological areas with lowered pH values can be visualized with pH-responsive contrast liposomes through MRI. In vivo tissue pharmacokinetics of nanoliposomal drugs in mice was monitored. Diethylenetriamine- Penta acetic acid liposomes were recommended for cancer therapy [68]. Radiofrequency tumor extermination by intravenous liposomal doxorubicin resulted in enhanced tumor aggregation of liposomes and improved necrosis in the tumors [69]. The copper-liganded bioactive complex was loaded in the new polymeric nanoliposomes with pH-responsiveness were designed and prepared for a controlled drug delivery system for the treatment of inflammation. The results show the high oxidant/ inflammatory inhibitory action of the prepared copper-liganded bioactive complex [Copper-glyglycineprednisolone succinate] ([(Cu(glygly)(PS)]) in comparison to the free PS-drug. Results propose that the new copperliganded bioactive delivery system with the controlled release of drug can function as a possible drug delivery system for the treatment of inflammation [70]. 2.3. Gold Nanoliposomes Gold nanoparticles and conjugates of nanoparticles with DNA extracted from cancer cells of human breast were prepared. Nanoparticles were blended with nanoliposomal hydroxyurea. Cytotoxicity on MCF-7 cells was measured by MTT assay and found that nano conjugated complex in concentrations less than 20 µM of hydroxyurea can expand efficacy compared with the liposomal drug [71]. Nanoliposomes were used as an ophthalmic carrier for nanogold topped with flucytosine drug. Gold nanoparticles were used as an antagonistic agent to trace the flucytosine in the later segment of the eye to treat fungal intraocular endophthalmitis. The computed tomography imaging technique was used to check the


ocular penetration of the nanoliposomes in vivo and found to be (10.22 ± 0.11 mm). The formulation has been found effective in treating the infected rabbits’ eyes [72]. 2.4. Nanoliposomes as Photosensitizing Agents Photodynamic Therapy (PDT) is a rapidly progressing technique in the management of many tumors, including skin tumors. Malignant cells are abolished by photosensitizing agents. Nanoliposomes functions as drug carriers and enhancers in PDT [73]. Using aloe emodin nanoliposomes, the collective consequences of gene transfection with PDT on gastric carcinoma cells were analyzed. The nanoliposomes functioned as a gene carrier and a photosensitizer. 77.3% increase in the death rate was observed after PDT (6.4 J/cm2) in the transfected cells. Nanoliposomes mediated gene transfection together with PDT could slow down the propagation rate and amplify the apoptotic rate exceptionally [74]. Miconazole Nitrate (MN)-loaded propylene glycol (PG) nanoliposomes gives controlled MN delivery, invariable PG uptake in the vesicles in the PG concentration range 2.510%, superior vesicle permanence, and superior MN skin deposition with lowest skin infiltration [75]. Hybrid encapsulation structures were developed based on β-carotene-loaded nanoliposomes incorporated within the polymeric ultrathin fibers produced through electrospinning improved the photostability of the antioxidant [76]. 2.5. Nanoliposomes as Peptides and Protein Carrier For topical and transdermal delivery peptides, vaccines and proteins, both in vitro and in vivo, elastic nanoliposomes have been developed. The lipid bilayer composition contains an edge activator to give flexibility. They are functional as non-occluded to the skin and have been demonstrated to penetrate all the way through the stratum corneum due to the hydration of the skin [77]. Integrity studies performed on nanoliposomes with ligands for Ab-peptides for Alzheimer's disease in the existence of serum proteins showed that these nanoliposomes achieve the conditions for in vivo applications. The binding capacity of the nanoliposomes was mediated by the existence of the tricyclic ligand on the surface. ThT assay of liposomes revealed a considerable reduction in thioflavin T fluorescence after 24h, signified a significant inhibition/delay of Ab1-42 aggregation [78]. Pediocin is a small antilisterial polypeptide bacteriocin was encapsulated in the nanoliposomal formulation. The effect of process criterion of the size of nanoliposomes was evaluated. With the increase in the concentration of phospholipids; particle size increases. However, the size decreases as the rise in amplitude and extent of sonication. Encapsulated pediocin was found to be more effective in impeding bacterial multiplication as compared to directly added pediocin. Hybrid capsules of alginate with guar gum integrated with pediocin-loaded nanoliposomes of phosphatidylcholine were originated to be one of the finest delivery systems for controlled release of pediocin [79]. Nanoliposomes consist of biologically active peptides with angiotensin-converting enzyme inhibitory and antioxidative and properties, derived from winged bean seeds protein was formulated. Winged bean seeds powder was papain-proteolyzed and encapsulated by the solvent-free heating method. Nanoliposomes confirmed excellent storage


67

stability for eight weeks at 4°C, determined by a minor increase (15.1%) in particle size and a decrease in zeta potential by 17.2%. These results suggested the possibility of entrapping water-soluble peptides in a hydrophobic nanoliposomal system that, upon optimization, has the perspective to act as a biologically active food component [80]. Navitoclax (Nav) can distinctively encourage apoptosis in Cancer-Associated Fibroblasts (CAFs). Navitoclax loaded nanoliposomes customized with peptide FH (FH-SSL-Nav), which particularly binds to tenascin-C, a protein mostly expressed by CAFs, were formulated. As compared to SSLNav, FH-SSL-Nav attained superior cellular uptake and stronger cytotoxicity in vitro. FH-SSL-Nav has a better tumor eradication potential by abolishing CAFs in Hep G2 tumor-bearing nude mice model [81]. The encapsulation of the peptides increases their delivery, stability, and bioavailability. The effect of the different range of molecular weight of peptides on their encapsulation efficiency within the nanoliposomes derived from soy lecithin was studied. The liposome ζ-potential is dependent on the molecular weight of peptide, which signifies that the charged groups of peptide have different positions relative to the surfaces of the liposome. The outcomes recommended that the peptides were unevenly dispersed within the liposomes, even with the similar encapsulation efficiency. These outcomes are significant for designing delivery systems for marketable production of encapsulated peptides with enhanced functional attributes [82]. Liposomes are being used as vesicular containers, especially for hemoglobin (oxygen therapeutic) as a blood substitute. The spatial separation of hemoglobin by a double layer of a lipid reduces the hemodynamic effects related to customized forms of hemoglobin. Liposomal hemoglobin exhibited a circulation half-life up to 65h for PEGylated liposomal hemoglobin formulation; this indicates the physiological durability [83]. Heat Shock Proteins (HSPs) are members of several families of stress-induced proteins, whose main intracellular functions are as molecular chaperones [84]. The HSPs possess the intrinsic property of recognizing unfolded /disordered sequences in target polypeptides, then aiding the folding/refolding of such sequences, targeting them to the proteasome for destruction. In addition, after exiting the cell and entering the extracellular environment, HSPs can promote antigen presentation of chaperoned peptides through interaction with the receptors on antigen presenting cells [85]. The Heat-Shock Protein-Peptide Complex (HSP.PCTu) derived from the tumor is a promising antitumor agent. An improved HSP70. The PC-based vaccine was refined from the Dendritic Cell (DC)-tumor fusion cells (HSP70. PC-Fc) which improved immunogenicity due to superior antigenic tumor peptides compared to HSP70.PC-Tu. In this study, the peptide complex was encapsulated with nanoliposomes (NL-HSP70. PC-Fc) to upsurge the bioavailability of HSP70.PC-Fc. The results obtained for NLHSP70.PC-Fc, which increases the bioavailability and improved immunogenicity of HSP70.PC represents superior Heat Shock Proteins (HSPs)-based tumor vaccines [86]. Encapsulation of active proteins in the hydrophilic core of vesicular liposomes is significant for developing a therapeutic


protein delivery system. The effects of operating parameters such as phospholipid concentration, buffer pH, ionic strength, protein size, surface charge, and liposome size on the enzyme encapsulation yield were investigated. The electrostatic interaction between the phospholipid and enzyme was the most significant parameter in determining the encapsulation yield. Thus adjusting buffer pH and ionic strength and adding charged phospholipids to the liposome preparation to impart electric charge to the lipid bilayer could improve the yield [87]. HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cell), a molecular complex of human α-lactalbumin and oleic acid, has selective cytotoxic activity against certain types of tumors. This cytotoxicity is known to stem from water-insoluble oleic acid. Nanolipoplex LIMLET (LIposome Made LEthal to Tumor cell) was manufactured. LIMLET showed distinctive cytotoxicity against A549 and MDA-MB-231 cells, while bare liposomes (containing no oleic acid) had no toxicity, even at high concentrations. The strength of the tumoricidal effect appeared to stem from the number of oleic acid molecules present [88]. 2.6. Nanoliposomal Vaccines Nanoliposomal vaccine delivery system uses the cellassault capacity of viral capsule proteins. Fusogenic viral envelope proteins were used to customize the nanoliposome exterior shell [89]. Nanoliposomal formulations against ricin toxin, Yersinia pestis, and Ebola Zaire virus are in preclinical studies [90]. The combined therapy of iontophoresis and nanoliposomes for transdermal drug delivery provided encouraging outcomes [91]. Non-lipidized recombinant Borrelia burgdorferi vaccine was formulated. OspC protein was anchored by metal chelating bond onto the surface of nanoliposomes which contains nonpyrogenic lipophilized norAbuMDP analogues denoted MT05 and MT06. After immunization, the experimental vaccines surpassed alum regarding OspC-specific titers of IgG2b isotypes, IgG2a when MT06 was used and IgM isotypes, IgG3 when MT05 was used. Both adjuvants employed a better adjuvant result than MDP and established themselves as nonpyrogenic [92]. The monoclonal antibody was conjugated with apoB-100 to the liposomal surface to prepare the immunoliposomes by using the post-insertion method. Results show that the intravenous injection of an only dose of apoB-targeted anionic nanoliposomes increases the lipid profile of serum. These outcomes have inferences for the management of patients with statin intolerance or severe dyslipidemias [93]. 2.7. Nanoliposomes as the Gene Delivery System Ultrasound-responsive liposomes consist of a little quantity of gas. Co-encapsulation of the drug along with the gas makes the liposomes dynamic in nature against sound for controlled release of the loaded drug [94]. Gene delivery using ultrasound and nanobubbles is accepted as a perfect system to transport plasmid DNA noninvasively into a particular target position. Bubble liposomes (BLs)/lipid nanobubbles of perfluoro propane were prepared for contrast enhancement. BLs induced cavitation when exposed to ultrasound and could transport plasmid DNA into the cells in vitro and in vivo [95]. To evaluate the effectiveness of a neu-

Dahiya and Dureja

tral 1,2-dioleoylsn-glycero-3-phosphatidylcholine (DOPC) nanoliposome system for the delivery of siRNA to tumor cells in a murine cervical cancer model was formulated. The formulation showed 65% and 57% decrease in tumor volume and weight respectively [96]. Folate-nanoliposomes have been used to deliver plasmids and siRNAs. A bone marrow and bone metastasis xenograft mouse model was established by inserting the LA-N-5 cell into the bone marrow cavity. The folate-nanoliposomes trapped MYCN siRNA can be distinctively circulated in tumor tissues. Studies show that folate-nanoliposomes ensnared MYCN siRNA led to MYCN mRNA expression extensively down-regulated as compared to negative control siRNA treatment. MYCN protein manifestation was repressed about 60% in vivo, therefore, induced tumor cell apoptosis distinctly [91]. A siRNA against the transcription factor E2F1 (siE2F1) loaded nanoliposomes were produced through a dedicated ultrasound assisted technique producing particles with about 40 nm size (Small Unilamellar Vesicles, SUVs) and 100% siRNA encapsulation efficiency. The study concluded that the siE2F1-SUVs generated to have the potential to contribute towards the development of novel effective inflammatory bowel disease-associated colorectal cancer therapies for a future personalized medicine [92]. 2.8. Chemotherapeutic Applications The targeting ability of activated carbon nanoparticles (DTX-AC-NPs) and nanoliposomes (DTX-LPs) was compared, to deliver docetaxel (DTX) to the metastatic lymph nodes. DTX-AC-NPs and DTX-LPs may be utilized for in vivo lymph node targeting. DTX-AC-NPs improved DTXAUC(0-24) and provide long-lasting DTX-retention in lymph nodes than DTX-LPs [93]. Mitoxantrone was encapsulated in liposomes comprising the Short-Chain Sphingolipid (SCS), C8-Glucosylceramide or C8-Galactosylceramide in their bilayer, to decrease the toxicity and to enhance mitoxantrone tumor cell accessibility. Liposomal encapsulation lessens the mitoxantrone toxicity with increased clearance and minor skin accumulation. The targeting of plasma membranes with SCS enhanced mitoxantrone tumor accessibility and thus therapeutic action and depicts an encouraging perspective to improve mitoxantrone-based chemotherapy [94]. Nanoliposomes of methyl 6-methoxy-3-(4-methoxyphenyl)1H-indole-2-carboxylate, was assessed for the in vitro cell growth inhibition on three human tumor cell lines of melanoma, breast adenocarcinoma, and non-small cell lung cancer. After an uninterrupted exposure of 48h showed very low GI50 values for all the cell lines tested (0.25 to 0.33 μM) [95]. Lactoferrin nanoliposomes have anticarcinogenic, antioxidant and antibacterial properties. The methyl thiazolyl tetrazolium assay established that lactoferrin nanoliposomes and lactoferrin activated in the cells in a way of dose-effect relation and lactoferrin nanoliposomes had a significant difference (p