1 Virus-like nanoparticles. 2 Cationic carriers for gene delivery

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1 This lecture highlights the strategies that have been evolved to use biomimetic materials as gene delivery vehicles such that they exhibit virus-like efficiency in delivery of the oligonucleotides. 1 Virus-like nanoparticles Virus-like nanoparticles are synthetic molecules that can interact with oligonucleotides to form complexes that possess the ability to deliver the oligonucleotide at a specific site similar to virions. These virus-like nanoparticles are also referred to as non-viral vectors. The complexes formed using lipid-based carrier molecules are referred to as lipoplexes and those formed with polymeric complexes are referred to as polyplexes. The following sections detail some of the major virus-like nanoparticle systems that have been investigated over the past few years. 2 Cationic carriers for gene delivery One of the main challenges in encapsulating oligonucleotides into a carrier is the huge amount of electrostatic repulsion that is encountered due to the phosphate backbone of the oligonucleotides. Hence, presence of a cationic component is essential to stabilize the oligonucleotide and carrier complex. Greater the number of positive charges, better will be the complexation of the negatively charged oligonucleotide with the carrier. Hence, delivery systems with high density of positive charges are preferred for gene delivery applications. Many factors influence the efficiency of the complexation as well as the particle size and stability. The number of positive charges on the cationic carrier will determine the rate at which the complexation occurs. Also, higher number of positive charges will enable more number of oligonucleotides to be complexed in a single particle. The type of oligonucleotide will also influence the complexation efficiency. Oligonucleotides can be either double stranded, single stranded, supercoiled or in the form of a circular plasmid. Figure 1 shows the different forms of oligonucleotides that can be used for gene therapy. Joint Initiative of IITs and IISc Funded by MHRD Page 3 of 11

2 Fig. 1: Different forms of oligonucleotides used in gene therapy The efficiency of complexation is superior for oligonucleotides with lesser number of base pairs.the size of the carrier-oligonucleotide complex depends on the molecular weight of the oligonucleotide, which in turn depends on the number of base pairs in the oligonucleotide. Higher number of base pairs generally tends to increase the particle size of the complex. It is interesting to note that when oligonucleotide complexation occurs with the cationic components in the carrier, the size of the complex decreases due to compaction driven by the electrostatic forces of interactions between the anionic oligonucleotide and the cationic carrier. How can one confirm that complexation has occurred? Well, an agarose gel electrophoresis can answer that question. On complexation with a carrier, linear DNA will be compacted into a spherical compartment and hence the shape factor is expected to contribute to faster mobility in the gel under the influence of an electric field when compared with uncomplexed linear DNA. Similar observations have also been observed with complexes with plasmid. However, the surface charge on the complex as well as its molecular weight will also influence the mobility of the oligonucleotide complex in an electric field. While free DNA is negatively charged, during complexation with the cationic carrier, there will be neutralization of charges. Also, higher efficiency of complexation will result in a substantial increase in the molecular weight of the complex. The combination of these two factors will retard the mobility of the carrierr and in many cases the complex might not move from the well. This is the case when small interference RNA (si-rna) about base pairs in size is complexed. The complex migrates minimally when compared with the free si-rna in an applied electric field. In the case of linear DNA-cationic carrier complexes and plasmid DNA cationic carrier complexes where the number of base pairs is generally greater than 100, the compaction to a spherical shape contributes majorly to the mobility under applied electric field. During complexation, a slight excess of the negative charges on the surface due to the DNA or plasmid DNA is present. This coupled with the spherical compacted complex formed, enables the complex to move faster when compared with its uncomplexed counterpart in an applied electric field. Incidentally, the presence of a surface charge on the complex Joint Initiative of IITs and IISc Funded by MHRD Page 4 of 11

3 also contributes to its colloidal stability as it prevents aggregation through electrostatic repulsive forces. The presence of negatively charged oligonucleotides and cationic carrier in a system facilitates fast association in a cooperative manner. The cationic amphiphile selfassembles to a micellar structure using the oligonucleotide as template. This association occurs above a critical concentration known as critical association concentration (CAC). Lower CAC values imply highly cooperative interactions between the carrier and the oligonucleotide and results in high complexation efficiency. Thus a carrier containing a high density of positive charges that can effectively complex with all the negative charges in the oligonucleotide is preferable. How can one be sure of arriving at the correct quantities of cationic charges required for perfect complexation? A quantifiable measure will be the N/P ratio. This ratio is based on the number of amino groups (N) from the cationic carrier to the number of phosphate groups (P)in the oligonucleotide. Ratios greater than 2 is generally required for effective complexation. Values greater than 4 has been found to be great for complete complexation and cell entry, but unfortunately, the cytotoxicity of the complex also increases due to the abundant positive charges. Another important factor that influences the transfection efficiency is the route of internalization of the carrier-oligonucleotide complex. This will decide the intracellular barriers such as lysosomal degradation, residence time in the cytoplasm, elimination through efflux systems etc. Apart from this, in the case of plasmid DNA, the nuclear localization remains a huge challenge. Generally, fast-dividing cells favour transfer of the plasmid DNA into the nucleus during the mitosis. But in the case of slow dividing cells, the percentage of plasmid DNA transferred into the nucleus is negligible resulting in poor transfection efficiency.hence, identifying the correct combination of the carrier and oligonucleotide with all qualities to ensure effective site-specific delivery is more like a tightrope walk and needs extensive experiments to arrive at the right balance. In the following sections, let us look into several types of cationic carriers that have found favour as gene delivery systems. It should however be noted that though each type have their own merits, they also possess certain demerits. 2.1 Poly(ethylene imine) delivery systems Poly(ethylene imine) or PEI is a cationic polymer that contains secondary amine groups. Figure 2 gives the structure of PEI. Joint Initiative of IITs and IISc Funded by MHRD Page 5 of 11

4 Fig. 2: Structure of poly(ethylene imine) The molecular weight of the polymer determines the number of positive charges it can have. The polymer can effectively complex with the negatively charged oligonucleotides due to its cationic nature thus making it an excellent choice for gene delivery systems. However, apart from its excellent oligonucleotide complexation properties, PEI also demonstrates another important characteristic that makes it invaluable in the field of gene delivery applications. The presence of secondary amine groups in PEI make it weakly basic. If it enters the endosome, the weakly basic nature of PEI tends to neutralize the acidic ph of the endosome (the ph of the early endosome is between while that of the late endosome is below 6). To offset the increase in ph, the ATPase-dependent vacuole proton pump (V-ATPase) is activated to pump in protons into the endosome. This proton influx also causes an influx of chloride ions (for electrical neutrality) and water molecules. This changes the osmotic pressure leading to swelling of the cell and finally disruption of the endosomal membrane causing the release of its contents in to the cytoplasm. This mechanism is now known as the proton sponge effect and offers a unique method for the nanoparticles to escape from the endosome. Figure 3 depicts the sequence of events that occur during the proton sponge mechanism triggered by the entry of PEI complexes in to the endosome. Joint Initiative of IITs and IISc Funded by MHRD Page 6 of 11

5 (A) Proton pump (B) Proton pump ph acidic ph increases PEI Endosome Endosome (C) Proton influx (D) Swelling occurs PEI Endosome Endosome disrupted with release of PEI complexes Fig. 3: Events occurring during the proton sponge mechanism Interestingly, no known virus uses the proton sponge mechanism for escape from the endosome! Maybe their current mode of endosomal escape is even superior than the proton sponge mechanism and hence they do not adopt this strategy! If it is true that the weakly basic nature of PEI is responsible for the proton sponge effect, then a similar effect must also be observed with other weak bases. Interestingly, chloroquine, an anti-malarial drug that is also weakly basic has been found to exhibit a similar disruption of the endosomes and the even more acidic lysosomes. Hence, it is referred to as a lysoosmotropic agent. How can one be sure that the activation of the V-ATPase proton pump is the cause for endosomal membrane disruption by PEI or chloroquine? Well, experiments carried out in the presence of bafilomycin, a known inhibitor of V-ATPase proton pump showed that PEI was unable to escape from the endosome in the presence of bafilomycin. This proves that the activation of the V-APTase proton pumpis essential for release from the endosome by disruption of its membrane. Thus, the PEI polymer possesses both complexation as well as endosomal escape properties that can be exploited for gene delivery. Joint Initiative of IITs and IISc Funded by MHRD Page 7 of 11

6 How does the PEI-oligonucleotide complex enter a cell? The cationic particles can bind through electrostatic interactions to transmembrane glycoproteins called syndecans, which contain heparansulphate and chondroitin sulphate moieties that make them anionic. This binding triggers clustering of the syndecan-bound PEI-oligonucleotide complexes into lipid rafts, leading to activation of protein kinase C mediated phosphorylation of tyrosine residues present in the associated proteins in the intracellular domain. This initiates a signaling cascade leading to actin polymerization and invagination of the membrane to form a vesicle with the help of dynamin protein. The same sequence of events can be witnessed if the syndecan bound PEI-oligonucleotide complex is transferred into caveolae. Once inside the cell, if the vesicle fuses with an endosome, the PEI-oligonucleotide complex will release from the endosome due to the proton sponge effect. However, due to the cationic charges present in the PEI polymer, it can bind easily to the negatively charged surface of the cell membrane through electrostatic interactions and can enter in to the cells. The electrostatic interactions can disrupt the cell membrane architecture leading to cell death by necrosis. On the other hand, if the polymer gets inside the cell and exhibits similar electrostatic interactions with the negatively charged mitochondrial membrane, this can lead to disruption of the mitochondrial membrane causing the release of pro-apoptotic factors resulting in the onset of programmed cell death (apoptosis). In either case, the cell death is more probable, thus making poly(ethylene imine) as a highly cytotoxic polymer. Another facet to PEI-based delivery systems is the possibility that the cationic PEI can bind to the anionic components in the extracellular matrix (ECM) resulting in nonspecific effects.hence, strategies to mask the surplus positive charges in PEI might provide a potential solution. In order to avoid non-specific entry of PEI into other cells leading to side effects, it is essential to modify the surface of PEI with a targeting ligand that will interact with a specific receptor on the cell of interest. This will ensure that PEIoligonucleotide complexes mostly will enter the target cells by receptor-mediated endocytosis. 2.2 Cationic lipid-based carriers for gene delivery Cationic lipid-based systems remain a widely investigated area for gene delivery applications. One of the commonly used nanostructures is the liposome, especially the small unilamellar vesicle (SUV). The advantage of a liposomal delivery system is the ease of preparation by self-assembly, the ease of surface modification with groups/molecules to impart specific properties such as long circulation, site-specific entry, fusion enhancers etc. The complex formed between a cationic lipid and oligonucleotide is known as a lipoplex. Commonly employed cationic lipids are DOTMA (N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride), DOTAP (N-[1-(2,3- Joint Initiative of IITs and IISc Funded by MHRD Page 8 of 11

7 dioleoyloxy)propyl]-n,n,n-trimethylammonium chloride) and DOGS (dioctadecylaminoglycylcarboxyspermine). Figure 4 gives the structure of these cationic lipids. DOTMA DOTAP DOGS Fig. 4: Structure of some popular cationic lipids used in gene delivery systems (DOTMA (N-[1-(2,3- dioleyloxy)propyl]-n,n,n-trimethylammonium chloride), DOTAP (N-[1-(2,3-dioleoyloxy)propyl]-N,N,Ntrimethylammonium chloride) and DOGS (dioctadecylaminoglycylcarboxyspermine) These structures are amphipathic containing highly hydrophobic acyl chain and a polar head group containing the positive charges. This resembles the phospholipid structure and hence when introduced into an aqueous environment self-assembles to a vesicular structure. The cell entry is facilitated by fusion with the anionic syndecans on the cell membrane surface. However, it is important to ensure that the lipoplex escapes from the endosome in order to have a chance to deliver the oligonucleotide to the intended target site (cytosol in the case of si-rna and nucleus in the case of plasmid). Hence it is necessary to introduce a component that can aid endosomal escape. Dioleoylphosphatidylethanolamine (DOPE) is an amphipathic phospholipid which when introduced into a lipid carrier can help in endosomal escape by disrupting the membrane. The structure of DOPE is given in Figure 5. Joint Initiative of IITs and IISc Funded by MHRD Page 9 of 11

8 NPTEL Nanotechnology - Nanob Nanobiotechnology Fig. 5: Structure of DOPE Being a phospholipid, it can easily integrate with lipid-based based carrier systems. How does incorporation of DOPE promote endosomal escape? The oleic acid moiety in DOPE has a double bond that is the cis isomer. This means that similar groups are on the same side of the double bond. This results in the bulky acyl domains occupying one side of the double bond and the hydrogen atoms the other side. As a consequence, close packing of neighboring lipid molecules is hindered. Also, the polar head group of DOPE is small and hence the their hydration volume also is small. Due to its small polar head group and oleic acid acyl chains, DOPE is not cylindrical but rather conical. When a liposomal bilayer is formed using DOPE as one of the constituent lipid, then a thin layer of water occupies th thee interstitial regions formed between the conical DOPE molecules. This exposes the hydrophobic acyl chains to water a condition that is not preferable. As a result, the DOPE components try to transform to another more stable phase rathe rather than exist in thee lamellar phase. In the endosome, the acidic ph enables protonation of the amino group in the ethanolamine moiety of DOPE. This enhances electrostatic attractive forces between the cationic amine group and the anionic phosphate group in the phospholipids. These forces along with the presence of unfavourable water layer drive the formation of DOPE-rich rich domains where the DOPE molecules tend to adopt an inverted hexagonal phase. Figure 6 shows the structure of a lamellar lipid bilayer and inverted nverted hexagonal pphase transition undergoneby the DOPE. Lipid bilayer Inverted hexagonal Fig. 6:: Inverted hexagonal phase transition in DOPE Joint Initiative of IITs and IISc Funded by MHRD Page 10 of 11

9 This lamellar to inverted hexagonal phase transition not only destabilizes the vesicular architecture but also binds to the endosomal membrane thereby leading to its perturbation and disruption. This enables the release of the endosomal contents into the cytosol. As DOPE alone could not form stable vesicles due to its instability in the lamellar phase, it is usually introduced as a component in lipid carriers that can form vesicles. However, even small quantities of DOPE in the carrier can promote endosomal perturbation aiding in the release of the carrier into the cytosol. Hence, DOPE is referred to as a helper lipid. Thus cationic lipids along with DOPE will be a good combination to achieve sufficient complexation of oligonucleotides as well as escape the lysosomal degradation pathway. One of the key properties that is required for effective lamellar to inverted hexagonal phase transition is the ease with which the lipid carrier membrane curvature can be altered. In other words, presence of membrane rigidifying components such as cholesterol can delay the transformation and hence affect the efficiency of endosomal escape. Too much DOPE will also reduce the membrane stability and hence its composition has to be tightly regulated to achieve the correct balance between a stable lipid carrier with optimal complexation potential and capability to escape from the endosome. A major concern in this type of carriers is the toxicity associated with the cationic lipids as well as the stability of the lipid carrier during circulation. A major chunk of research is directed towards developing or modifying cationic lipids that exhibit lesser toxicity, a discussion of which by itself requires a separate chapter! In the following section we will look at some special surfactant-based systems that have immense potential in gene delivery. 3 Reference Smart Nanoparticles in Nanomedicine (The MML series, Vol. 8), Editors: Reza Arshady& Kenji Kono, Kentus Books, 2006 Joint Initiative of IITs and IISc Funded by MHRD Page 11 of 11