Novel Non-Viral Gene Carrier System: Recent Progress and Current Status
Manju Rawat, D. Singh, Swarnlata Saraf and S. Saraf*,
Institute of Pharmacy, Pt. Ravishankar Shukla University,Raipur 492 010 (C.G.)
INDIA
ABSTRACT
Safe, efficient and specific delivery of therapeutic genes remains an important
bottleneck for the development of gene therapy. The limitations of viral vectors,
in particular their relative small capacity for therapeutic DNA, safety concerns,
difficulty in targeting to specific cell types have led to evaluation and development
of alternative vectors based on synthetic, non-viral systems. Synthetic, non-viral
systems have already been established and showed a unique pharmaceutical profile
with potential advantages for certain applications.
The incorporation of new design criteria has led to recent advances toward
functional delivery systems. This review focuses on the mechanistic aspects
of non-viral vectors such as cationic lipids, cationic polymers protein and
peptide, lipids, liposomes etc. delivery agents and novel method using vector
systems. It can control gene expression especially for genes whose therapeutic
effects are considerably dependent on quantity, site, duration and timing eg.
Temperature responsive vector system. The genesis of various agents is not here
discussed but this review discusses recent progress in various segments of synthetic
polymers that have recently been explored as delivery vehicles, focusing not
only on their strengths, but also on their limitations.
INTRODUCTION
The safe and efficient delivery of therapeutic DNA to cells represent fundamental
obstacle to clinical success of gene therapy [1,2]. Today's gene therapy has
gone beyond the original definition of gene therapy [3]. Gene therapy has given
an opportunity to fight the cause of a disease rather than its symptoms. Now
more than 45,000 human diseases have been identified related directly to the
genetic disorders [4]. With the advent of gene manipulation by biotechnological
techniques it has now become feasible to splice and insert human gene into viral
or bacterial genome while the latter is referred as vector. The technique is
essentially based on recombinant DNA technology which allows the isolation of
genes and their subsequent utilization in the production of respective proteins,
as well to engineer them to be a corrective gene system.
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In principle, two basic carrier systems, viral (adeno and retrovirus) and
non-viral for DNA delivery in target cells are under development. Viral vectors
were most commonly used in gene therapy clinical trials because of their high
transfection efficiency, but their efficiency of transfection in vitro
is not reproduced in vivo because of their inherent inflammatory properties,
coupled with inappropriate tropisms, which restrict their access to target tissues
[5]. The concept of using block or graft copolymers of cationic and hydrophobic
nonionic monomers has been introduced as a potential means for development of
non viral gene delivery vectors [6]. The characteristics of viral and non-viral
vector systems are summarized in Table 1.
Table 1 : Characteristics of viral and non-viral vector system [68]
| Vectors | Characteristics | Disadvantages |
| 1. Viral vector system | ·Relatively high titers. ·Stable gene expression due to viral genome integration into cell chromosomes. ·Can infect non-dividing cells. ·Total insert capacity in the virion is in the range of ·Transiently high levels of gene expression. | ·Host immune responses, inflammatory and toxic ·Random insertion of viral genome, which may possibly result in mutagenesis. ·Possibility of replication competent virus formation by homologous |
| 2. Non-viral vector system | ·They aren't infectious. ·Theoretically, there is no limit to the size of DNA. ·They are suitable for oligonucleotide delivery. ·Low degree of toxicity | ·Targeting isn't specific. ·Low transfection efficiency. ·Only transient expression. ·Difficult in vivo applications. ·Host immune responses, inflammatory reactions in patients if they |
NON-VIRAL GENE CARRIER
Non-viral gene carriers are often used cationic lipids and polymers [7,8].
A complex is formed between anionic DNA and cationic lipid or polymer by ionic
interactions. This complex is formed with excess positive charge. Therefore
it could interact with the anionic cell membrane to induce endocytosis of the
complex in a highly efficient manner. Generally, cationic carrier-DNA complex
accomplishes transcription of the therapeutic gene by the following five steps:
(1) The complex adheres to the cell surface by electrostatic interactions
(2) Cellular endocytosis occurs into endosomes.
(3)The complex translocates from the endosomes to the cytoplasm by mechanisms
such as lipid fusion [9] and proton sponge effect [10,11].
(4)The complex or the released DNA from the complex moves into the nucleus
(5)Lastly the transcription process is initiated.
In non-viral DNA delivery no single formulation can be used to target all
somatic targets. The formulations need to be optimized for each somatic target
on the basis of physiological and biological characteristics.
There are varieties of non-viral gene carrier system have been developed such
as liposomes ,cationic lipids and polymers ,stimuli responsive polymers, peptides,etc
1. Liposomes
Liposomes are microscopic spheres composed of one or more closed, concentric
phospholipid bilayer membranes surrounding an internal aqueous compartment[12].
Negatively charged or classical liposomes have been used to deliver encapsulated
drugs and act as carrier. Due to the Problems associated with the efficiency
of nucleic acid encapsulation and complexes from "ghost" vehicles leads to the
development of positively charged liposomes. This positively charged liposomes
prepared with cationic lipids which is able to interact spontaneously with negatively
charged DNA to form clusters of aggregated vesicles along the nucleic acid which
works as a carrier for DNA.
2. Cationic Lipids
The cationic lipid as such work as carrier for DNA by complexing with nucleic
acid of DNA. There are three common types of cationic lipids and are employed
in lipid-based DNA delivery [14]. The first group is represented by two quaternary
ammonium salts with long mono-unsaturated aliphatic chains, eg. N-(2,3(dioleyloxy)
propyl)-N, N, N trimethyl ammonium chloride (DOTMA, one component of the commercially
available transfection agent LipofectinÒ) and N, N-dioleyl-N,N-dimethyl
ammonium chloride (DODAC).Second group includes 3b-[N-(N-(dimethylamino ethane)
carbamoyl] cholesterol 9DC-Chol, a cationic derivative of cholesterol. Lipids
of a third category are distinguished by the presence of multivalent headgroups,
such as dioctadecyldimethyl ammonium chloride (DOGS), commercially available
as TransfectamÒ.
Fabrication of carrier system
The DNA or oligonucleotides when mixed with lipids, it forms complexes spontaneously
due to electrostatic interactions. These electrostatic interactions result in
condensation of polyanionic nucleic acid molecule into a tightly packed structure
[15]. These structures work as carrier systems. Generally smaller particles
are preferred for both in vitro and in vivo applications [16].
The size of carrier system is dependent on the size of initial lipid vesicles,
the positive to negative charge ratio, the ionic strength of the medium and
the concentration of all reagents. Recent data of Ross [17] demonstrated that
the efficiency of transfection with cationic liposomes-DNA complexes (lipoplexes)
increased with increasing lipoplex size. Moreover, by using large lipoplexes,
the degradative properties of serum may be minimized, when complexed with lipids,
DNA is protected from nulcease degradation to various extents [18].
The delivery of the complexes of DNA or oligonucleotide by lipids into the
cell cytoplasm occurs by the endocytotic-lysosomal pathway and not by direct
delivery through the fusion of lipid-DNA carrier system with the plasma membrane
[19,20,21]. Lipid mixing in lipid-DNA particles and the endosomal membrane is
an important part of the mechanism of endosomal release [22]. Lipids capable
of promoting intermembrane lipid exchange (e.g. unsaturated PEs, unsaturated
acyl chains and hydroxylated headgroups) increase the ability of lipids to disrupt
the endosomal membrane [23]. Dioleoylphosphatidyl ethanolamine [DOPE] is particularly
useful for cationic liposomes because of its ability to form non-bilayer phases
and promote destabilization of the bilayer [24].
Fig. 1 : Proposed mechanism of internalization into cells of cationic lipoplexes
and release of DNA into cytoplasm [70]
Zelphati and Szoka proposed a model that describes oligonucleotide release
from the endosomes Fig1. After the cationic lipid – nucleic acid complex is
internalized by endocytosis, it destabilizes the endosomal membrane. This destabilization
induces a flip-flop between anionic and cationic lipids. The neucleic acid is
displaced from the cationic lipid and diffuses into the cytoplasm of the cell.
The DNA/cationic lipid complex is transferred to lysosomes and the nucleic acid
may be rapidly degraded by nucleases. Alternatively, full-length oligonucleotides
are released from endosomes into the extracellular compartment by exocytosis
[71]. The release from the endosomes and subsequent diffusion into the nucleus
represents the rate-limiting step for DNA transfection. Nuclear pores allow
the molecules of the size of anti-sense oligonucleotides to diffuse freely between
the nucleus and cytoplasm. Cationic lipid vesicles not only enhance the rate
of uptake into cells but also markedly change the sub-cellular distribution
of the oligonucleotide. The major difference in the distribution of the oligonucleotide
in the presence of cationic lipids is the localization of the oligonucleotide
in the cell nucleus [20].
However, cationic lipids as oligonucleotide carriers have several disadvantages.
The main disadvantages of cationic lipids are their toxicity and markedly decreased
activity in the presence of serum [25]. Newer cationic lipid formulations are
available that exhibit decreased toxicity [26, 27, 28].e.g. The inclusion of
a helper lipid (DOPE or cholesterol)in new formulation reduces the effective
charge ratio required to deliver oligonucleotides into cells and permits delivery
in the presence of high serum concentration[25]. The amount of serum protein,
bind to liposome/DNA complexes can be reduced by increasing the dose of liposome/DNA
complex administered intravenously. This reduction in protein binding produced
a corresponding increase in circulation times (4 minutes to over 80 minutes.)
However, polymeric gene carriers have been studied because of some advantanges
over the lipid systems:
(1)Relatively small size and narrow distribution of complex [13].
(2) High stability against nucleases; and
(3) Easy control of physical factors (e.g. hydrophilicity and charge) by
co-polymerization
3. Cationic Polymers (Cps)
The cationic polymer utilizes for gene delivery known as a dendrimer. Dendrimer-DNA
complexes are formed by ionic interactions between the positively charged dendrimer
and negatively charged DNA. These complexes effect efficient gene delivery into
a variety of cell types in vitro [40]. Like cationic lipids. Polymers
bearing groups that are protonated at physiological pH have been employed as
gene carriers. The electrostatic attraction between the cationic charge on the
polymer and the negatively charged DNA results in a polyplex. Table 2 represents
the chemical structures and some molecular characteristics of cationic polymers,
frequently used as gene carriers.
TABLE - 2A : Cationic homopolymers as gene carriers
Some CPs, such as PLL (poly L-lysin) are linear polymers while other one like
PEI (polyethylenamine) and dendrimers are highly branched chains. The block
copolymers like the PEG-PLL and comb type copolymers like PLL-gr-dextran with
polycation backbones and grafted hydrophilic side chains DEAE-dextran considered
as predecessor of the CPs for gene transfection [30]. But it has low transfection
efficiency, toxicity and non-biodegradability [29, 49]. The linear PLL has chain
length heterogeneity that results major variabilities in size distribution of
polyplexes which mainly based on oligolysines and synthetic polypeptides [31,32].
Cationic copolymers bearing hydrophilic segments (PEG) improve solubility and
stability of polyplexes [33-40]. A new class of cationic polymers as candidates
for gene carriers with transfection properties is pAMAM dendrimers [40].
More recently, methacrylate based CPs [41, 42] and cationic polysaccharides
like chitosan [43] have been introduced in studies on gene carriers. The targeting
of gene complexes to a desired cell population is an important subject in the
field of gene therapy. Many CPs can be easily conjugated to targeting ligands.
Among them PLL has been the most widely used for attaching targeting ligands.
Recent publications have reviewed ligand-PLL systems in detail [44, 45, 49].
TABLE -2B : Cationic copolymers studied as gene carriers
TABLE 3 : considers the principal ligands used to target
PLL polyplexes and updates ligands studied in combination with other CPs.
Table 3
| CP | Target Cell | Ligand | Ref |
| pLL | Hepatocytes Hepatocytes Hepatocytes Macrophages Lung epithelial cells Various cell types | Asialoorosomucoid Lactose, galactose Insulin based ligand Mannose Fab fragment of IgG Transferrin | 29,47,46 48,50,51 52 53 54 55, 56 |
| PEI | Hepatocytes | Galactose | 72 |
| Trimethyl chitosan | Hepatocytes | Galactose | 73 |
4. Stimuli-Responsive Polymers
Synthetic stimuli(temprature) responsive polymers can be applied in gene carrier
system.It is also known as intelligent polymers Cationic gene carrier systems
have a common dilemma in such systems. It must fulfill the following two opposite
requirements simultaneously (Fig.) for carrier system of gene.
(1)Tight complex formation, favorable for cell uptake and evasion of DNA degradation.
(2)Complex dissociation or loose complex formation, favorable for transcription
by RNA polymerase.
Light sensitive polymers have advantage over temperature sensitive polymers
with respect to site precision, however, temperature can also be applied to
a specific site with considerable precision, such as within 5 mm using an ultrasound
device [57]. Most widely studied temperature responsive polymer is poly (N-isopropyl-acrylamide)
PIPAAm, which is known to exhibit a temperature-dependent phase transition behaviour
with a lower critical solution temperature (LCST) at 32oC [58, 59,
60] It has extensive biomedical applications as hydrogel [61,62], bioconjugates
[64] and polymeric micelles as gene carriers [64-66].
Fig. 2 : The dilemma of the DNA-polymer complex
Fig. 3 : The concept of temperature-responsive gene carriers
Below the LCST, PIAAm is water soluble and hydrophilic and exist in extended
chain form. Above the LCST, PIPAAm undergoes a reversible phase transition to
an insoluble and hydrophobic aggregate. Using this properties of polymer, a
complex between the carrier polymer and DNA can be tightly formed above the
transition temperature by hydrophobic aggregation of polymer (Fig. 3).and complex
as DNA carrier.
Therefore, a change between the tight complex formation and its dissociation
(or loose complex formation) can contribute to optimize efficiency of gene delivery
and consequently, the total gene expression efficiency could be dramatically
increased with the potential for selective gene expression.Other thermally responsive
block copolymer micelles comprising poly(N-isopropylacrylamide b-DL-lactide)have
been studied extensively.Block copolymers of poly(N-isopropylacrylamide) and
poly (butylmethacrylate) have also been worked upon[65, 66].
Peptide as Gene Carrier
In 1946, Kleczkowski investigated [67] that the conjugation of proteins with
nucleic acids is a general phenomenon that takes place whenever the pH allows
them to be of opposite charges and also suggested that the interaction of proteins
with viral nucleoproteins are responsible for reducing the infectivity of some
viruses.On the basis of these observations the approach of using proteins for
DNA delivery is based on. The functionally active regions of proteins such as
enzymes, receptors and antibodies having relatively small, typically consisting
of around 10-20 amino acids are selected for gene carrier. Synthesizing peptides
based upon functional regions of DNA binding proteins or a variety of viral
proteins is an approach that use to replace the use of whole proteins (such
as histone H1) or large polydispersed polymers (such as polylysine)
as gene delivery. A number of peptide sequences are able to bind to and condense
DNA. One such sequence is the tetra-peptide serine-proline-lysine-lysine located
in the C-terminus of the histone H1 protein. Rational design of peptide
sequence can be used to develop completely synthetic DNA binding peptideswhich
act as carrier.
Albumin is another protein that can interact with DNA complexes by electrostatic
or hydrophobic interactions. Albumin interacts with positively charged complexes
and form ternary albumin -DNA-polycation complexes. Some parameters of the DNA
complexes, such as Z-potential or hydrodynamic radius when change it effectively
acts as a carrier for DNA.
CONCLUSION
This review has focused on the various synthetic, non viral systems utilizing
various polymers including cationic polymers, cationic lipids, stimuli responsive
polymers and include liposomes, liposome-polycation complexes and peptide delivery
systems.
The improvement of vector designs using various polymers has allowed for a
broader range of therapeutic applications for gene transfer technology. Gene
therapy has a more active role in clinical trials and there has been a dramatic
increase in the number of preclinical studies for gene therapy and genetic immunization
programs however, the paradigm for the development of polymeric gene delivery
vectors remains the incorporation of these design elements into materials as
part of an interactive, linear process an effective albeit slow, approach to
the discovery of new vectors.
Currently a series of chemically different CPs, ranging from linear homopolymers
to block and comb type copolymers has been reported to condense DNA and proposed
as gene carriers. Cationic lipids have also been extensively studied as an effective
agent for DNA transfer in vivo. Several different cationic lipids have
been used to deliver genes to the lung by intratracheal, intravenous (IV) or
intrapulmonary artery routes resulting in efficient expression of recombinant
genes. Gene delivery with cationic liposomes increases the level of gene expression
intravenously as the lipoplex prevent plasma degradation of DNA. Compared with
the high efficiency of viral gene transfection, the efficiency of gene transfection
in vitro and especially in vivo by polyplexes and lipoplexes is still
relatively low. Rational designing of available polymers and lipids needs to
be done, attention should be paid to all critical steps in the process of lipoplex
and polyplex transport from extracellular space into the nucleus considering
the critical importance and complexity of DNA dissociation from lipoplexes and
polyplexes, there is currently an urgent need for advanced physicochemical methods
which allow characterizing these critical steps in such media. In order to overcome
shortcoming of above discussed vector system stimuli responsive vector
system has been worked upon to control gene expression. Gene expression control
by using temperature sensitive polymers is one of the easiest and safest signals
applied to living bodies this synthetic vecor system could find applications
for in vivo gene therapies. Another novel class of delivery agents based on
several types of proteins and peptides which have the ability to penetrate cell
membranes have been developed. Rational design of peptide sequence can develop
completely synthetic DNA binding peptides.
In summary, the number of potential synthetic polymer based gene delivery systems
has expanded tremendously over the past several years. However, it isn't clear
which of many currently available technology provides optimal delivery, defined
as maximal activity for the least unit cost. Moreover, fundamental research
on the toxicological and immunological aspects of polymer based delivery system
is highly recommended as the information currently available is limited. Undoubtedly,
these are some of the many challenging problems in this area that will occupy
numerous scientists over the next decade.
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*Author for correspondence
Prof. S. Saraf has nearly 17 years of research and teaching experience at both U.G. and P.G. levels. He is a leading scientist and well-known academician . Prof. Saraf did his doctoral research at the Dept. of Pharmacy, Dr. H. S. Gour University, SAGAR. under the supervision of Prof. V. K. Dixit, a renowned Pharmacognosist. He has over 50 research publications to his credit published in international and national journals. He has delivered invited lectures and chaired many sessions in several National Conferences and Symposia in India. His research interest extends from Herbal Cosmetics to Herbal drug standardization Modern analytical techniques, New Drug Delivery Systems with biotechnology bias. He has authored 1 books, in press. Presently, he is Professor and Director Institute of pharmacy and Dean, Faculty of Technology, Pt. Ravishankar Shukla University , Raipur , (C.G.)
corresponding address
*S. Saraf
Professor & Director
Institute of Pharmacy
Pt. Ravishankar Shukla University
Raipur, 492 010 C.G.
0771-2263773 - O&Fax
Email: shailendrasaraf@rediffmail.com
Dr. (Mrs.) Swarnlata Saraf has nearly 14 years of research and teaching experience.
She is a leading scientist and well-known in the field of herbal cosmatics.
Mrs. Saraf did her doctoral research at the Dept. of Pharmacy, Dr. H. S. Gour
University, SAGAR. She has over 40 publications to her credit published in international
and national journals. She is an active member of IPA ,APTI and ISTE. Her research
interest extends from Herbal Cosmetics to transdermal drug delivery (specially
Iontophoresis), New Drug Delivery Systems for biological and therapeutic agents.
She had Co-authored 1 books, (in press). Presently, She is working as a Reader
at Institute of Pharmacy Pt. Ravishankar Shukla University, Raipur, (C.G.) INDIA.
Mr. Deependra Singh has nearly 6 years of research and teaching experience.
He is a researcher with a different vision. Mr. Singh did his masters degree
from Dept. of Pharmacy, Dr. H. S. Gour University, SAGAR. He has over 16 publications
to his credit published in international and national journals. He is founder
secretary of IPA local branch Chhattisgarh. His research interest extends from
Noble topical delivery systems, Delivery Systems for biologicals to Plant tissue
culture . Presently, he is working as a Lecturer at Institute of pharmacy Pt.
Ravishankar Shukla University, Raipur, (C.G.) INDIA
Ms. Manju Rawat has 3 years of research and teaching experience. She has been
a bright student through out. Ms Rawat did her M.Pharm from Dept. of Pharmacy,
Dr. H. S. Gour University, SAGAR. She has about a dozen research publications
to her credit published in international and national journals. Her research interest
extends from Protein delivery to novel Drug Delivery. Presently, she is working
as a Lecturer in Pharmacy Institution in INDIA.