Lipid- and polymer-based plexes as therapeutic carriers for bioactive molecules

Accepted Manuscript Review Lipid- and polymer-based plexes as therapeutic carriers for bioactive molecules Pravin Shende, Narayan Ture, R.S. Gaud, F. Trotta PII: DOI: Reference:

S0378-5173(19)30023-7 https://doi.org/10.1016/j.ijpharm.2018.12.085 IJP 18057

To appear in:

International Journal of Pharmaceutics

Received Date: Revised Date: Accepted Date:

16 November 2018 25 December 2018 27 December 2018

Please cite this article as: P. Shende, N. Ture, R.S. Gaud, F. Trotta, Lipid- and polymer-based plexes as therapeutic carriers for bioactive molecules, International Journal of Pharmaceutics (2019), doi: https://doi.org/10.1016/ j.ijpharm.2018.12.085

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Lipid- and polymer-based plexes as therapeutic carriers for bioactive molecules Pravin Shende1*, Narayan Ture1, R.S. Gaud1 and F. Trotta2 1

Shobhaben Pratapbhai Patel School of Pharmacy and Technology Management, SVKM’S NMIMS, V. L Mehta Road, Vile Parle (W), Mumbai, India. 2

Department of Chemistry, University of Torino, Italy.

Address for correspondence Dr. Pravin Shende Shobhaben Pratapbhai Patel School of Pharmacy and Technology Management, SVKM’S NMIMS, V. L Mehta Road, Vile Parle (W), Mumbai, India. Tel No. +91-22-42332000 Fax No. +91-22-26185422 Email. [email protected] 1

Abbreviations DOPE

Dioleoyl phosphatidyl ethanolamine

DC-chol

Dimethyl amino ethanol carbamoyl cholesterol

DOPC

Dioleoyl phosphateidyl choline

DOTMA

(N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride)

DODAB

Dioctadecyl dimethylammonium bromide

DODAC

Dioctadecyl dimethylammonium chloride

DDAB

Dimethyloctadecylammoniumbromide

DMRI

Dimyristooxypropyldimethyl hydroxyethyl ammonium bromide

DOGS

Dioctadecylamidoglicylspermin

PEI

Poly(ethylenimine)

PLL

Poly(L-lysine)

BGSC

Bis-guanidinium-spermidine-cholesterol

PAMAM

Polyamidoamine

pHis

Homopolymer of histidine

PEAA

Polyethyl-acrylic acid

PAGA

poly[alpha-(4-aminobutyl)-L-glycolic acid

GDNF

Glial cell-derived neurotrophic factor

VEGF

Vascular endothelial growth factor

AT2- R

Angiotensin type 2 receptor

IL

Interleukin

2

Graphical abstract

3

Abstract Recently, promising strategies of plexes include the complexation of nucleic acids with lipids (lipoplexes) and different kinds of polymers (polyplexes) for delivery of actives and genetic material in abnormal conditions like cancer, cystic fibrosis and genetic disorders. The present review article focuses on the comparative aspects of lipoplexes and polyplexes associated with molecular structure, cellular transportation and formulation aspects. The major advantages of lipoplexes and polyplexes over conventional liposomes involve nonimmunogenic viral gene transfer, facile manufacturing and preservation of genetic material encapsulated within the nanocarriers. Lipoplexes and polyplexes enhance the transfection of DNA into the cell by stepwise electrostatic cationic-anionic interaction with DNA backbones. The ease and cost-effective formation of complexes extend their applications in the treatment of cancer and genetic disorders. Lipoplexes and polyplexes necessitate intensive research in the fields of quality, toxicity and methods of preparation for commercialization.

Keywords: Nanoparticulate drug-delivery systems; lipid; polymer; lipopolyplexes

4

1. Introduction Nanoparticulate drug delivery systems (NPDDSs) including nanodroplets, nanospheres, nanocapsules, nanosponges and liposomes, are sized in a nanodimensional range from 1 to 100 nm and offer an improvement in the therapeutic efficacy of drug by modulating its release and stability, prolonging circulation time and protecting from elimination of phagocytic cells or premature degradation. Among various nanoengineered systems, the site-specific targeted systems involve polymeric micelles, dendrimers, polymeric and ceramic nanoparticles, metal nanoparticles, quantum dots (QDs), dendrimers, viral-derived capsid nanoparticles, polyplexes and liposomes (Conti et al., 2006). These nanocarriers show advantages in the treatment of cancer, genetic diseases, most of inherited and acquired diseases and cystic fibrosis mostly due to increase in drug solubility and stability, improve pharmacokinetic property and enhance permeability and retention (EPR) effect-mediated tumour targeting. Presently lipoplexes and polyplexes are the areas of interest due to complexation phenomenon of these carriers with DNA (Jafari et al., 2012). In the lipid-based delivery system, DNA can be surrounded by lipids in an organized structure like a micelle or liposome whereas lipid complexed DNA is called a lipoplex (Tros de Ilarduya et al., 2010). The most widely used applications of lipoplexes in gene transfer for cancer treatment by modulating activated genes to tumor suppressor control genes in the cell and decrease the action of oncogenes. Lipoplexes enhance the transfection of DNA into the cell by electrostatic interaction between cationic lipid head groups and the phosphate DNA backbones. The lipoplexes are effectively taken up by cells and produced cytotoxicity and apoptosis at the site of action. In case of polyplexes, the complexation of polymer with DNA occurs and mostly initiated using cationic polymer. The plasmid DNA is shielded by polyplexes and lipoplexes by blocking the entry of nucleolytic enzyme while plain DNA is degraded rapidly. The differences between lipoplex and polyplex delivery systems are shown in table 1. 5

Table 1. Difference between lipoplex and polyplex systems Lipoplexes

Polyplexes

Present in the form of complexes of lipid Present in the form of complexes of polymer and DNA.

and DNA.

Lipoplexes conatining the cationic lipids In polyplexes, endosomal release becomes destabilize

the

endosomes

by

direct more complicated.

interaction with the membrane. (Tros de Ilarduya et al., 2010). They transfer from endosome to cytosol by They transfer from endosome to cytosol by ‘flip-flop theory’. (Zelphati and Szoka, ‘proton sponge effect theory’. (Vermeulen et 1996).

al., 2018).

Lipoplexes discharge their DNA load into Polyplexes cannot discharge their DNA load the cytoplasm.

into the cytoplasm.

Lipoplexes are present in lamellar Lα phase Polyplexes are typically toroidal or spherical and inverted hexagonal phase.(Tros de structures. Ilarduya et al., 2010). E.g. (N-[1-(2,3-dioleyloxy) propyl]-N, N, N- E.g. Poly(L-lysine) (PLL) trimethylammonium chloride) (DOTMA), Poly(ethylenimine) (PEI) and Chitosan Dioleoylphosphatidylethanolamine (DOPE) and Dimethylaminoethanol-carbamoyl Cholesterol (DC-chol).

Both lipoplexes and polyplexes are non-immunogenic in nature, in contrast to viral gene carriers and show a capacity to protect the genetic material from degradation. Cationic lipoplexes are more toxic than anionic and neutral complexes. Cationic lipoplexes easily form 6

complex with negatively charged genetic materials while neutral and anionic lipoplexes are difficult to undergo complexation. However, the comparative low transfection efficiency is the main drawback of such types of systems. Polyplexes offer effective complexation of polymeric materials with genetic materials. The disadvantages include toxicity and nonbiodegradability of polymers like PEI. Extensive studies illustrated that the polyplexes form complexes with drugs to enhance their solubility, retard the degradation and offer sitespecific drug delivery.

These versatile polymeric structures demonstrate the ability to

combine various types of polymers like PEI and PLL for controlled release of drug. By optimizing the polymer composition and modifying surface characteristics, such nanocarriers provide significant advantages for the delivery of nucleic acids. 2. Formation of lipoplexes and polyplexes In comparison to viral gene delivery system, the transfection efficiency of novel cationic lipoplexes are enhanced, however, the mechanism of lipoplex formation is still under investigation. The formation of lipoplex involves electrostatic interaction between cationic charges of lipid and anionic charges of DNA. The negative charges of DNA are finally neutralised by surrounding a single plasmid by adequate cationic lipids that can deliver net positive charge which is then interact with cell surface for transfection of lipoplex in cell. The factors like concentration of lipid, temperature, environmental condition and kinetics of mixing play important role for lipoplex formulation (Ma et al., 2007). The mechanism of formation of lipoplex is shown in figure 1.

7

Figure 1. Mechanism of formation of lipoplex

The formation of lipoplexes occurs by mainly two processes: DNA-induced membrane fusion and liposomes-induced DNA collapse. The later involves vesicle formation and attachment of DNA within the ring-like lipid bilayer of liposomes. A quasi-spherical compact complex of DNA and lipid developed during the process of complexation with a particle diameter of approximately 0.2 mm. A neutral complex consists of string-like colloidal complexes with highly ordered multi-lamellar internal structure. In the preparation of polyplexes, transfection efficiency and stability of polyplexes are affected by the selection of polymer. Polyplex formulation is carried out depending upon its ionic qualities and the relationship between polycation/polyanion which is fast and relatively irreversible. The first step for the formulation of polyplexes is the complexation of DNA with polymer which affects the polyplex size and its transfection efficiency (Gebhart and Kabanov, 2001). The polymer properties like molecular weight, molecular number, charge density and the DNA-polymer ratio influence condensation and complexation behaviour. The condensation of PEI with plasmid DNA or RNA results in the formation of a stable complex because of electrostatic interactions where PEI acted as polymer of choice for endosomal escape (Tang and Szoka, 1997). The mechanism of formation of polyplex is shown in figure 2.

Figure 2. The mechanism of formation of polyplex 8

3. Mechanism of action of lipoplexes and polyplexes The lipoplexes and polyplexes mediate gene transfection and demonstrate a notable progress in understanding the mechanisms of cellular pathway. The nanosized lipoplexes and polyplexes are formed from cationic liposomes and polymers which are further bound to nucleic acids (DNA or RNA). The steps involved in the mechanism of lipoplex and polyplex are described below: 3.1 Mode of action of lipoplex in the body cell Step I: Interaction of lipoplexes with the cell surface and their internalization into the cytoplasm with the help of non-specific electrostatic interaction and these particles bound to the cell surface (Friend et al., 1996). Lipoplexes enter the cell and interact with anionic proteoglycans by the transmembrane protein called syndecans. The lipoplexes mostly contain cationic complexes which are in bound forms with the negatively charged surface of cells. These resulted to endocytosis like mechanism and end with the transfer of the complex into the cell. Eukaryotic cell uses different endocytic pathways, like clathrin-mediated endocytosis, macropinocytosis and caveolae-mediated endocytosis. With the exception of uptake in particular cells like monocytes and neutrophils, macrophages avoided as a critical system of lipoplex uptake in a typical cell. The size of lipoplexes influences the process of internalization and effectiveness of transfection. In most of the cases, after adsorption on the cell surface, lipoplexes enter the cells through endocytic pathways and transported with microtubules to perinuclear area. Step II: Release of lipoplex cargo from endosome The bound part of nucleic acid breaks down from endosomes into the cytosol. DNA released from the surrounding of lipid to perform its biological functions. In the case of DNA-cationic lipid complex or lipoplex, disruption of the endosome occurs through the interaction with the 9

cationic lipid by trans-bilayer flip-flop of anionic lipids from the external layer of the endosomal membrane. It was found that DNA can be released in the case of complete neutralization of positive charge. Lipid composition of both lipoplexes and cellular membranes can stimulate the phase behaviour of lipid mixtures and can be responsible for DNA release. For DNA release into the surrounding space, the bilayer barrier should be disintegrated. After internalization into endosomes, lipoplexes exchange lipids mostly through interaction and fusion with the surrounding endosomal membrane. Endosomes containing lipoplexes interact with various cytoplasmic membranes and exchange lipids with them until complete neutralization of cationic lipid takes place, which is necessary for release of DNA. After the disintegration of lipoplexes, confocal microscopy reveals a broad distribution of cationic lipids and DNA in the cytoplasm, while a minimal concentration of DNA and lipid enter in the nucleus (Midoux et al., 2008). Step III: Routes of DNA penetration into the nucleus There are two pathways for release of DNA from lipoplexes and polyplexes: DNA enters the nucleus by passive transport during cell division when nuclear membrane breaks down temporarily or by active transport of DNA through nuclear pores. Nuclear pores seem to be the most probable gate for DNA penetration into the nucleus. Other nucleus composed of a few thousand pores constituted of nuclear pore complex (NPC) proteins (Doye and Hurt, 1997). The basic mechanism for cellular transportation of lipoplex is shown in figure 3.

10

Figure 3. The basic cellular transportation mechanism of lipoplex

3.2 Mode of action of polyplexes in the body cell Cationic polymers are deficient of hydrophobic group and freely soluble in water, unlike cationic lipid. These are easily synthesized with different modifications in structure and length. Hence various structure-function relationship studies are based on the different stages of transfection.

Step I: Cell binding and uptake

The successful transfection efficiency depends on the polyplex internalization into cells and type of polymer used. Polyplexes enter the cell and interact with anionic proteoglycans by using transmembrane protein called syndecans. The association of polyplexes to the cell and their uptake into cells are dependent on highly positive charge of surface of polyplexes. The transfection efficiency of the complex depends on the type of polymer used for the synthesis of complex. Linear PEI-polyplexes use a clathrin-coated pit pathway for transfection in hepatocellular carcinoma cells (HUH-7) and in African green monkey kidney (COS-7) (von Gersdorff et al., 2006). Both lipid raft and clathrin dependent pathways are used by branched 11

polyplexes such as in HeLa cells, but the lipid-based pathway is more efficient in the transfection process.

Step II: Escape from endosome PEI and PAMAM-based polyplexes indicate high transfection efficiency as they acted on proton sponge. Proton sponge hypothesis states that when polyplexes enter the cells through the endocytosis, they reside in endosomal vesicles. Upon maturation, the membrane-bound V-ATPase proton pumps actively translocate protons into the endosomal lumen. Since the polymers used in the proton sponge hypothesis shows a high buffer capacity, they are able to bind these protons, thereby limiting the acidification of endosome. As a result, the proton pumps will translocate even more protons to the endosomal compartment in an attempt to lower the pH of the environment. The translocation of protons is accompanied by the entry of chloride ions (to maintain the charge balance) which lead to an increase in ionic concentration and influx of water to maintain osmolarity. The influx of water molecules generates an osmotic pressure that leads to swelling of the endosomes and polymer due to internal charge repulsion, eventually causes endosomal rupture with release of endosomal content into the cytosol (Godbey et al., 2000). A recent study proposed that “proton sponge” mechanism is utilised to achieve high transfection efficiency in PEI-based polyplexes (Midoux et al., 2008). Step III: Dissociation of polyplexes

After the endosomal escape, polyplex must reach the nucleus where DNA dissociates from the carrier. Thus polyplexes of low molecular weight tend to dissociate rapidly and lead to better transfection as compared to high molecular weight polyplexes. PEI-based polyplexes show a slow rate of dissociation from DNA when analysed using fluorescence microscopy.

12

The intracellular condition of reducible PLL polymer is resulted into quick release and improves expression activity (Ogris et al., 2001).

Step IV: Nuclear import

Nuclear entry through passive process occurs when nuclear membrane disassembles during cell division. PEI shows an ability to facilitate nuclear translocation of DNA because microinjected PEI-DNA polyplexes which reveal higher transfection ability when compared to naked DNA or lipoplexes. The basic mechanism of transportation of polyplex is shown in figure 4.

Figure 4. The basic mechanism of transportation of polyplex 4. Toxicity of nanocarriers Lipoplexes and polyplexes are widely used as non-viral gene carriers. However, their applications are complex due to the acute inflammatory toxicity associated with their systemic administration. Several clinical and animal trials have demonstrated the toxic

13

behaviour of lipid-based gene carriers at high doses. Recently, several studies demonstrated the toxicity associated with DOTMA, DOTAP and DMRIE. In these studies, there are three types of toxicities observed due to high doses of lipid-based nanocarriers: inflammatory toxicity, hepatotoxicity and serological toxicity. The systemic administration of lipoplexes rapidly activates the innate immunity and symptoms like headache, rise in body temperature and myalgia are observed within 6 h of administration. The response is generated due to activation of inflammatory mediators like cytokines, interferons and interleukins. The activation of immune mediators is a function of complex between lipid and DNA. Lipid, when used alone, does not necessarily cause toxicity. Furthermore, the cytokine-mediated toxic reaction can be reduced by sequential administration of lipoplexes through intravenous route. PEGylation of nanocarriers is a promising approach applied to improve the pharmacokinetics characteristics of encapsulated drugs, and to achieve better therapeutic outcomes with fewer side effects. Liposomes, when PEGylated are administered repeatedly into the body which causes reduction in the circulation time. This induced phenomenon is called accelerated blood clearance (ABC) effect. After the first dose of PEGylated liposomes administered in the body, the innate immune system recognizes the system as foreign moiety and produces anti-PEG IgM antibodies against the liposomes. When the second dose of the same formulation is given, the already developed anti-PEG IgM antibodies immediately bind with the liposomes and activate the complementary immune mediators to remove the foreign substance from the body. This causes the accelerated clearance of PEGylated systems from the body (Li et al., 2015). Polyplexes are synthesized using cationic polymers like PEI, PAMAM, etc. and these polymers show the tendency to get deposited in different tissues and organs like lungs, liver and kidney. This deposition leads to toxicity if not excreted from the body within a stipulated time period. A recent study demonstrated that a novel dextran-based polyplex with efficient transfection shows mild toxicity in the muscle. This is not shown any

14

evidence of toxicity in kidneys or liver. In a nutshell, cationic lipoplexes are more toxic than cationic polyplexes (Eliyahu et al., 2006). 5. Methods for preparation of lipoplexes and polyplexes 5.1 Formation of lipoplex by direct mixing Liposomal suspension was prepared by mixture of DC cholesterol and DOPE in the ratio of 3:2, then DNA solution was added into liposomal suspension to form lipoplexes. It was found that direct mixing of liposome and DNA shows better yield compared to the addition of DNA into liposomal suspension with dilution method (Meisel and Gokel, 2016). 5.2 Formation of lipoplex by thin film hydration method Thin film hydration technique involves the formation of liposomes by complexation of cationic lipid and neutral lipid in the ratio of 1:1. This mixture was added in a round bottom flask containing chloroform: methanol in the ratio of 4:1 (v/v). The solvent was evaporated to obtain lipid film using rotary evaporator. Then the resulting lipid mixture was disseminated aqueous sucrose solution and extruded through polycarbonate membrane with 0.1 pore size. The addition of suitable quantity of DNA per liposomes resulted into formation of lipoplexes (Radwan Almofti et al., 2003). 5.3 Large scale preparation of lipoplexes For the preparation of lipoplexes, the DAC-30w was mixed in lipid/DNA ratio (w/w) of 4:1. The liposomal preparation was prepared by dispersing of sterile lyophilised DAC-30w in transfection medium and incubated for 30 min. The preparation was filtered using polycarbonate membrane in presence of peristaltic pump. The plasmid and DNA suspension were diluted with transfection medium. The DNA and lipid were mixed using a Y-shaped connector pump and this mixture was further lyophilised to obtain dried lipoplexes. The preparation of lipoplexes is shown in figure 5. 15

Figure 5. Preparation of lipoplexes

5.4 Preparation of polyplexes by microfluidics The polyplexes preparation includes loading of a polymer and a solution of nucleic acid at ratio 1:1 and diluted in 5% glucose solution in two different syringes. The tubing was preloaded with solutions and worked at different flow rates and both the pumps were started simultaneously. Polyplex solutions were collected and particle sizes of fractions were measured (Debus et al., 2012). 5.5 Preparation of lipoplexes by microfluidics DOPE and DOTAP were dissolved in ethanol and the ethanol-lipid solution was injected into the first inlet and an aqueous buffer into the second inlet of microfluidic mixer. During initial studies, the total flow rate (TFR) of aqueous buffer and lipid phase were varied from 0.5 to 2 mL/min and the flow rate ratio (FRR) of the solvent and aqueous phases was varied from 1:1 to 1:5. The mixing of two adjacent streams results in aqueous dispersions of liposome formulations, then collected from the outlet stream and dialysed over night against trisaminomethane buffer of pH 7.4 to remove any residual solvent. Lipoplexes was prepared 16

by diluting in SUV solution with Opti-MEM and incubated for 30-40 mins at room temperature. After incubation, plasmid DNA was added in Opti-MEM and mixed with liposome solution with incubation for 15 mins at room temperature.(Kastner et al., 2015) 5.6 Preparation of chitosan-DNA polyplexes Chitosan polyplexes are more effective than PEI polyplexes as they possess better transfection ability than PEI polyplexes. A study found that the level of transfection was comparatively high with chitosan of molecular weight 40 kDa or 84 kDa (Ishii et al., 2001) due to large size. Hence, chitosan-based polyplexes emerge as a novel nanocarrier for gene therapy. Chitosan-based polyplexes prepared by dispersing chitosan and DNA in sodium sulfate solution and by further mixing it at high speed and vortexed to obtain polyplex dispersion. (Hallaj-Nezhadi et al., 2011). 6. Different types of lipids in lipoplexes Hemisuccinate (CHEMS), neutral [e.g. 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and cholesterol different types of lipids used in the production of lipoplexes can be classified as cationic, anionic and neutral as shows in figure 6.

17

Figure 6. Classification of lipids 6.1 Cationic lipids Cationic lipids are amphiphilic molecules which contain positively charged head groups linked with hydrophobic domain via neutral lipids such as DOPE and cholesterol. Cholesterol-containing lipoplexes show better stability in physiological media, shields DNA from degradation and facilitates transfection. The linked moieties are used to stabilize formulation during manufacturing, storage and administration but they showed rapid degradation at desired site of action (endosome). Many cationic lipids such as O-(2R-1,2-diO-(1,9-Z-octadecadienyl)-glycerol)-3-N-(bis-2-aminoethyl)-carbamate

(BCAT)

facilitate

transfection of the molecule (Boomer et al., 2002). DOTAP is chemically unstable but biodegradable (Hirko et al., 2003) whereas DOTMA is chemically stable but nonbiodegradable. DOPE forms hexagonal phase structures at neutral pH and participates in bilayer formation. Lipofectin was used as a standard for testing transfection efficiency of other lipid containing lipoplexes and composed of DOTMA and DOPE. CTAB (Yang et al., 2013) and DDAB are also used as cationic lipids in the preparation of nanoliposomes and nanocarriers.

6.2 Neutral lipid In the formulation of cationic liposomes, neutral lipid played a role of assistant. Three neutral lipids are normally used like DOPE, DOPC and cholesterol. Cholesterol and its derivatives are used for higher transfection level in vivo whereas neutral lipid like DOPE is used to improve carrier efficiency of lipoplex. The neutral lipid DOPE in cationic lipoplex leads to higher transfection efficiencies as the conical structure of this lipid promotes the formation of inverted hexagonal structures that rapidly fuse with the endosomal lipid bilayer, independent 18

of charge, thus enabling cytoplasmic release of DNA. Substitution of DOPE in the lipoplexes improves efficiency of carrier (Du et al., 2014). 6.3 Anionic lipid Anionic lipids are considered as a promising tool for gene therapy because of their low cytotoxicity. 6.4 Ionizable lipid Ionizable lipids are used in gene therapy due to the ability of self-assembling into nanoparticles when mixed with nucleic acid (polyanionic). Such lipids increase not only the payload of nucleic acid but also the efficiency of gene therapy. When pH is greater than pKa of ionizable lipids, neutral charge on nanoparticles evade RES uptake of lipids, thus improving the blood circulation along with reduced toxicity (Tam et al., 2013). In contrast to this, a lower pH results in protonation of anionic charges on lipids thereby facilitating the escape from endosomes. Two formulations of ionisable lipids i.e. DLin-MC3-DMA (1,2dilinoleyloxy-N,N-dimethyl-3-aminopropane) and DLin-KC2-DMA (2,2-dilin-oleyl-4-(2dimethylaminoethyl)-[1,3]-dioxolane) were prepared leading to improvement in the potency ten times more than that of conventional lipids with reduced half-maximum effective dose (ED50) ( Jayaraman et al., 2012; Heyes et al., 2005). Different lipids with their structures are shown in table 2.

Table 2. Types of lipids and their structures 19

Lipids

Structure

Description

Referenc es

DOTMA

DOTMA encourages both an uptake (Trancha and the expression of DNA in the tissue nt et al., cells and is mainly depends on the 2004) combination between complexes and plasma membrane.

DOTAP

DOTAP is extraordinary form compared to other lipids and generally utilizes as cationic lipid. It is moderately cheap, and effective in both in vitro and in vivo applications.

CHEMS

Its ability to settle PE in bilayer (Lai et membrane and to keep the development al., 1985) of hexagonal stage.

Cholesterol

Cholesterol settles the complex and (Hirschdrags out its blood time and is Lerner et exceptionally effective for delivery of al., 2005) nucleic acids into different cell composes in vitro and in vivo.

BGSC

Cholesterol with guanidinium polar (Vignero head bunches are productive and helpful n et al., reagents for gene transfer move into a 1996) wide variety of mammalian cell lines.

(EvenChen and Barenhol z, 2000)

20

DODAB

DODAB is a cationic, bilayer-shaping engineered lipid with a high compound stability. Effective DODAB system is low cost with additional antimicrobial action.

(Mamizu ka, 2007)

7.Different types of polymers in polyplexes Polymeric carriers are regarded as major non-viral alternatives for gene delivery due to their low immunogenicity and pathogenicity. The polymers comprise of linear branched and dendritic structures which display properties like flexibility in order to maintain the biocompatibility, facile manufacturing, robustness and stability of formulations utilised for efficient gene delivery. Recently, different types of polymers developed for the effective delivery of bioactives not only show less toxicity but also involve in various stages of delivery, for instance, escape of bioactive from endocytic vesicles into the cytoplasm. Many of such materials are better than off-the-shelf polymers as their delivery efficiency remains much below than that of recombinant viruses. Cationic substances which include amine-based polymers like chitosan, polylysine (PLL), polyamidoamine (PAMAM) and poly (ethyleneimine), etc., are employed as gene carriers. Polyplexes remain stable in enzymatic environment and improve the cellular entry of the complexes. Various polymers with their applications are tabulated (table 3) below: Table 3. List of polymers used in polyplexes Polymers

Structure

Descriptions

References

Polylysine

PLL is biodegradable peptide and (Zauner

(PLL)

mostly used in delivery of bioactive al., 1998)

et

substance. The transfection efficiency of polyplexes of PLL is lacked

21

because

they

trapped

after

endocytosis. Polyethylenei

PEI shows higher efficiency of (Ulasov

mine (PEI)

transfection

with

et

better al., 2011)

biocompatibility and less toxicity at a molecular weight of 25 kDa. Higher molecular weight, PEI shows greater transfection but promotes damage of cell membrane and apoptosis.

Chitosan

Chitosan contains huge amount of (Saranya amine

and

hydroxyl

et

groups, al., 2011)

additionally and improve the gene transfer. It shows less toxicity, higher biocompatibility, less

biodegradability,

immunogenicity

and

better

antimicrobial effect. PAMAM

It was discovered that the sixth- (Rudolph et generation dendrimer was superior to al., 2000) bring down ages by ~10-folds. Because

of

its

generally

high

delivery of gene productivity and great

biocompatibility,

PAMAM

dendrimers are utilized in few in vivo gene delivery studies. G-pHis

G-pHis shows greater transfection (Carrier, efficiency than PEI polymer. G-pHis 2003) is reported to be very less toxic in the delivery of bioactives like plasmid DNA, siRNA, etc.

PEAA

The hydrophilic nature of PEAA (Thomas

et

22

polymer transforms into hydrophobic al., 1994) as endosomes create partitions and destroy vesicle membrane during protonation in acidic conditions.

Cyclodextrin

The particle used in cyclodextrin (Hwang

polymers

polymers is modified by inclusion al., 2001)

et

complex. Combination of inclusion complex and off-the-shelf polymers is employed for the delivery of bioactive. PAGA

The transfection efficiency of PAGA (Lim et al., is 3-fold better than polylysine in the 2000) presence of chloroquine. PAGA exhibits

action

without

toxicity

unlike polylysine which decreases cell viability by 80%.

8. Characterization of lipoplexes and polyplexes Lipoplexes and polyplexes are characterised by various parameters as shown in table 4. Table 4. Characterization parameters with instruments Instruments

Parameters

Ideal characteristics

Transmission

To characterize the structure of Lipoplexes

electron microscope

lipoplex and polyplex

(TEM)

show

inverted

hexagonal

structure

whereas

polyplexes

are

toroidal

structure. Evaporative light

To determine the concentration The concentration of liposome

scattering

of lipoplexes and polyplexes

DNA combination is 4:1 23

detectors (ELS) FTIR spectroscopy

To measure the molar ratio of Determination cationic lipid and polymers

CIDIQ

of

functional

groups

To quantify the distribution of Distribution of exogenous DNA DNAs

in endosome/lysosome, cytosol, and nucleus to be quantified

Scanning electron

To characterize the lipoplex and Inverted hexagonal structure

microscope (SEM)

polyplex morphology

DLS

For the measurement of zeta -30mV to + 30mV is considered potential

of

lipoplex

and to

polyplex

be

an

ideal

range

for

detection

Confocal laser

To identify the fluorescently Determination

scanning microscopy

labelled

(CLSM)

polyplexes

Electron spin

To

resonance (ESR)

structure-based features of lipids bilayer

lipoplexes

determine

and escape

of

of

endosomal

lipoplexes

and

polyplexes motion

and Determination

and polymers

changes

properties

in and

incorporation of DNA

9. Applications of lipoplexes and polyplexes 9.1. Lipoplexes and polyplexes for anti-cancer therapy Non-viral gene therapy is an emerging and developing field in cancer therapy wherein specific and successful delivery of particular genes to target cells is possible. In contrast to viral vectors, non-viral vectors are attractive gene carriers in gene therapy because of their biosafety and capability to get chemically modified. 9.1.1. Drug-nucleic acid complexes Drug and nucleic acid complexes display tremendous potential in cancer treatment by affecting the pathways of different disease condition. Modern nanocarriers such as polyplexes, liposomes, nanoparticles and micelles are employed to enhance the efficacy of nucleic acid-drug complexes in disease conditions. Nucleic acid therapies such as plasmid 24

DNA (pDNA), small interfering RNA (siRNA), small hairpin RNA (shRNA), antisense oligonucleotides (AON) extend great attention. In the treatment of cancer bio-distribution and pharmacokinetics of drugs and nucleic acids are mainly affected by the differences in properties like molecular weight, susceptibility of compounds to biotransformation and hydrophobicity leading to fluctuation in drug concentration at the targeted site. Lipoplexes show strong cytotoxic and apoptotic activities because of the property of enhancing the chemo-sensitivity of malignant cells leading to decrease in the transfer of such cells. Predominantly, dioleoyl phosphatidylethanolamine (DOPE) and cholesterol are utilized in polyplexes formation to enable drug solubilisation and membrane fusion. In the delivery of Pgp siRNA and doxorubicin combination, polyplexs comprising DOPE and PEI have been used in the treatment of breast cancer (Biswas and Ilarduya, n.d.). Minko et al. prepared a single cationic liposome by using two transporters of siRNAs i.e MRP1 and Bcl-2 for delivery of doxorubicin (Saad and Minko, 2008). This system shows suppression of both the drug-efflux pump resistance and non-pump anti-apoptotic resistance resulting in enhanced anti-cancer efficacy. Lipoplexes and polyplexes are quite favourable as non-viral carriers for nucleic acid delivery into cells, for example, siRNA is employed for gene suppression and plasmids for transfection. Different nucleic acids, drugs and their combinations used for cancer therapy are shown in table 5. 9.1.2. Gene-nucleic acid complexes Lipoplexes and polyplexes are used as carriers for nucleic acid and effective in the treatment for genetic diseases. Preclinical development and recent advances in lipoplexes and polyplexes for delivery of nucleic acid are discussed in table 5. Table 5. Various nucleic acids, drugs and combination therapies used in cancer treatment

25

Nucleic

Delivery type

Drug/Gene

acid

Type

of Target

Reference

study Drug nucleic acid complex

Bcl-2

Lipoplex

5-Fluorouracil

In vivo

Colorectal

(Nakamura

cancer

et al., 2009)

Hepatic

(Cao et al.,

cancer

2011)

Multidrug

(Saad

2/MRP1

resistance

Minko,

SiRNA

cancer

2008)

Prostate

(Chen et al.,

cancer

2012)

Ovarian

(Sher et al.,

cancer

2012)

Breast

(Xiong

cancer

Lavasanifar,

siRNA

Bcl-2-

Polyplex

Doxorubicin

In vivo

siRNA Bcl-

Choline

Lipoplex

Polyplex

Doxorubicin

5-Fluorouracil

In vitro

In vivo

kinase

and

siRNA Endostati Lipoplex

5-Fluorouracil/

In vitro

n-yeast cisplatin

gene

MDR-

Polyplex

Doxorubicin

In vivo

siRNA

and

2011) Mcl1

Lipoplex

Paclitaxel

In vivo

siRNA

Nasopharyn

(Sung et al.,

x

2011)

carcinoma

Gene nucleic acid complex Plasmid

Intratracheal/

DNA

mice

AT2-R

Polyplexes

Lung cancer

(Kawabata et al., 2012)

26

siRNA

bi-

Intravenous

Intravenous

VEGF

KRAS

Polyplexes

Lipoplexes

shRNA Plasmid

Intratracheal/

DNA

mice

AT2-R

Polyplexes

Pancreatic

(Pittella

et

cancer

al., 2012)

Pancreatic

(Rao et al.,

cancer

2018)

Lung cancer

(Kawabata et al., 2012)

9.2. Lipoplexes and polyplexes as carriers in the treatment of various diseases Recently, nucleic acid-based actives extend great promise as a novel class of medicinal agents, but are restricted due to insufficient delivery at the target cells. Generally, lipoplexes and polyplexes are used to enhance cellular delivery of nucleic acid-based actives and their stability in bloodstream. Wide varieties of nucleic acid like small interfering RNA (siRNA), antisense oligodeoxynucleotides (AS-ODN), plasmid, DNA micro RNA (miRNA), etc., are utilized in the treatment of different diseases as shown in table 6. Table 6. Lipoplexes and polyplexes in the treatment of diseases Nucleic acid

Disease condition

Carrier system

References

Plasmid DNA

Cystic fibrosis

Lipoplexes

(Lindberg et al., 2012)

Plasmid DNA

Parkinson’s disease

Polyplex

(Huang et al., 2010)

SiRNA

Asthma

Polyplexes

(Xie et al., 2016)

pDNA

Haemophilia

Polyplexes

(Bowman et al., 2008)

SiRNA

Rheumatoid arthritis

Lipoplex

(Khoury et al., 2008)

10. Advances in nanocarrier systems for therapeutic delivery 10.1. Anti-cancer drug delivery and siRNA

27

Reports showed that both the nuclic acid and drugs were delivered to in vitro in the HeLa cells. In the tumour bearing Swiss Albino mice, decrease in the volume of tumour occurred after the administration of drug loaded polyplexes. Such demonstrates the therapeutic prospective of polyplexes in the delivery of doxorubicin and siRNA complex in cancer therapy (Nehate et al., 2017). Redox sensitive polyplex nano-system employed for the delivery of doxorubicin and kinase I siRNA. PEI modified copolymer poly [(poly (ethylene) glycol methacrylate]-polycaprolactone produced from ATRP in order to achieve better chemotherapeutic effect. These are applicable in vitro and in vivo studies and show high biocompatibility and haemocompatibility. 10.2. Delivery of self-replicating RNA vaccines by using polyplexes Polyplex-formulations display tremendous potential in the effective delivery of RepRNA to dendritic cells which facilitates cytosolic translocation for RepRNA translation. Humoral response and cellular immune response confirm the in vivo efficacy. The PEI-polyplexes showed the advantages like efficient delivery of complex and large self-amplifying RepRNA vaccines to dendritic cells. Overall, the cells present in intact form and delivery prompted RepRNA internalization by dendritic cells PEI-polyplexes can be considered as a reliable for all cells. Such differences are reflecting plasma membrane action and receptor ligation, or, in other words, cell subsets, with the receptors deciding the endocytic pathway for RepRNA delivery (Démoulins et al., 2015). 10.3. Lipopolyplexes in the treatment of Parkinson’s diseases Coupling of lipoplexes and polyplexes i.e. lipopolyplexes display advanced properties such as increase in colloidal stability, high transfection efficiency and reduction in cytotoxicity. Due to such properties, they are regarded as a next generation non-viral therapeutics. Lipopolyplexes show better gene packaging capability and lower immunogenicity. Chen et al. proposed that ‘‘Trojan Horse Liposome’’ (THL) is a system which is preferred over

28

bioactives for CNS treatments. In THL, an internal cavity of liposome condenses the DNA and protects it from degradation by nuclease enzyme. THL is set up by lipids which contain PEG in order to delay the blood circulation-time. Transferrin receptor mAB-focused on immunoliposomes for loading a tyrosine hydroxylase (TH) articulation plasmid to standardize the striatal TH movement (Chen et al., 2016). 10.4. PEGylated lipoplexes for ovarian cancer therapy Lipoplexes prepared using cationic lipid (DC-chol/DOPE) act as a carrier for the delivery of siRNA into cancerous cells (Shende and Patel, 2018). However, the negatively charged component of blood forms a complex with the bioactive. The PEGylation on the DCchol/DOPE liposomes is used in the formation of lipoplexes which enhance the delivery of bioactive substances and also improve systemic siRNA delivery in cancer therapy as proposed by Lee. Moreover, PEGylation helps to enhance the accumulation of active on the malignant cells, decrease the extraction by the kidneys and improve the circulation of lipoplexes in the bloodstream. The PEGylated lipoplexes complexed with siRNA used for the silencing of kinesin spindle protein (KSP) gene which shows high level of gene silencing at the site of cancerous cells, and significant destruction growth of tumor. Furthermore , in the immunocompetent mice, PEGylation lipoplexes did not

produce any innate immune

response (Lee and Ahn, 2018). 11. Conclusions Gene therapy mediated by lipoplexes and polyplexes are developed as an advanced alternative technique for viral delivery system. These therapeutic carriers impart advantages such as nonimmunogenic virogen transfer, ease of manufacturing and prevent degradation of genetic materials. Due to structural similarity, lipoplexes show better transfection efficiency as compared to polyplexes. The various aspects of plexes like quality, toxicity and methods of preparation require standard protocol for market approval. The future perspective of lipid-and 29

polymer-based plexes associated with nanotechnology an approach that seems to be quite favourable and significant in the treatment of various diseases. Conflict of interest Authors disclose no conflict of interest.

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