home

Vectors Viral Vectors Non Viral Vectors **Lipoplexes** Anionic and Neutral Liposomes Cationic Liposomes Introduction to Cyclodextrin PolyCationic Amphilic Cyclodextrin Derivatives for Non-Viral Gene Delivery Characterizing Ability of Amphiphilic Cyclodextrins for Non Viral Gene Delivery Cationic Amphiphilic Cyclodextrins Designed for Gene Delivery with Interesting Architectures and Synthesis **Assessment and Outlook for Poly cationic Amphiphilic Cyclodextrin Derivative as Non-Viral Vectors for Gene Delivery**
 * Gene Therapy**

Gene therapy is considered to be the therapy of the 21st century and has become a hot area of research because it aims to eliminate rather then symptoms of diseases.
 * Gene Therapy**

Gene therapy could be used to treat both genetic and non-genetic disorders. Genetic disorders caused by a mutation in a specific gene can be treated by delivering a copy of normal functioning DNA to the target cell or tissue to promote proper replications.1 For non-genetic disorders DNA can be delivered to the target to stimulate an immune response, whether that be producing genes that induce cell death, production of genes that will modify the cellular information or genes that produce therapeutic proteins with specific functions.2

Two different approaches have been developed for gene therapy; //**Ex vivo**// and //**In vivo**// gene therapy

//**Exo Vivo -**// In this approach specific cells are isolated and purified from a patient. The cells are then genetically modified and then re-infused to patient.3

//**In Vivo-**// This strategy involves direct gene transfer to tissue of patients either by delivery of Naked DNA or DNA in a vector.4



The goal of this strategy is to achieved expression of transgenes in target tissue for the require amount of time, in the appropriately regulated form and with no major side effects cause by interactions with the host genome.

The simplest method of gene therapy is injection of naked DNA plasmid, however this methods has major draw backs because of low transfection efficiencies and short term expression. Delivery and **Transfection** efficiencies can be increased by packed DNA in a carrier first, also known as a vector.

 Carriers of DNA, also known as vectors, have been developed to because of the low efficiency of injecting naked DNA. Vectors are meant to fulfill several functions such as:
 * Vectors**

a) Enable delivery of genes to target cell and nucleus b) Provide protection from degradation or immune attack c) Ensure gene transcription in cells

Vector should be inexpensive and easy to produce and purify in large amounts and concentrations. Vectors can be classified into two categories; **Viral** and **Non Viral** Vectors

Viral Delivery Vectors** Viral vectors are a common tool of Molecular Biologists. They use viral vectors to deliver genetic materials into cells. The process can be preformed inside a living organism //( in vivo//) or in cell cultures ( //in vitro//). Viruses have highly evolved specialized molecular mechanics to efficiently transport DNA to the cells they infect.
 * 

The transfection efficiencies of viral vectors are very high. The main disadvantage of using viruses as vehicles for gene delivery is their safety. Introducing a virus to the body can trigger an immune response which not only impedes the delivery of the genes to the target cells but can cause severe complications and side effects to the patient.

In 2002 viral vectors were used in gene therapy to treat rare immune disorders and initial results were promising. The excitement of the success of the study was soon trampled when the patients developed leukemia like symptoms. The viral vector was successful in inserting into the DNA to treat the disease however this ability was also responsible for activating a cancer promoting gene and causing the leukemia like symptoms.5

Defective virus can be used to reduce the risk of infection during handling or in gene delivery. Defective viruses are produced modifying the genome by removing the part of the viral genome that is responsible for viral replication. The virus is still able to infect the cell; however can not replicate unless the deleted portion is supplied by the cell line.

Interaction between vector and host cell genome cannot be completely eliminated and in the process of recombination the viral vector can recover the deleted portion of the viral genome and the virus if no longer defective. The virus that is now able to replicate is called a reactivant.

Although the chances of the viral vectors being converted back to its replicant competent form are low the results and risk from this occurring are so severe that the there are major safety concerns for using this for gene therapy**.**

Non-Viral Delivery Vectors**
 * 

The FDA (The US Food and Drug Administration) has not approved any viral-vector based therapeutics due to immunogencity, oncogenicity, and the possibility of viral recombination enabling replication of the virus. Non Viral vectors have gained increasing attention because of their relative safety and simplicity of use.

Of course, the simplest non viral gene delivery system is by injection of naked DNA. However the low transfection efficiencies of naked DNA and the safety concerns of viral vectors have pushed the development of non-viral vectors. In the past low transfection and expression were the main disadvantage of non viral vectors, however on going studies and development have produced non-viral vectors that have efficiencies comparable to viral vectors. There are several scaffolds that have been used for non-viral vectors, this discussion will focus on Lipoplexes.


 * 

Lipoplexes** Lipolex non-viral vector cover Plasmid DNA with lipids that organize into structures such as liposome and or micelles.

Lipids are composed of amphiphilic molecules that can organize to for well defined supramolecular structures held together by non covalent interactions. These compounds typically contain a hydrophobic head group and hydrophilic tail.


 * Amphiphile**

The head group can either be charged or uncharged. Charged head groups can be anionic and include the functional groups sulfates, carboxylates, sulfonates and phosphates. Positively charged head groups have a cationic amine functional group and non charged head groups are polar groups such as alcohols.

In aqueous solution the molecules will organize so that the hydrophilic groups will point towards the aqueous environment and the hydrophobic tail of the molecule will ‘hide’ from the aqueous solution and interact with other hydrophobic moieties. These interactions drive self assembly into larger supramolecular structures held together by non-covalent interactions. Depending on the shape and the hydrophobic/hydrophilic balance of the molecule different supramolecular structures can be formed. The below figure illustrate some of these structures.



The amphiphiles can arranges so that all the hydrophobic tails are pointed in and the hydrophilic head groups are point outward to form a sphere ( micelle) or a hexagonal columnar structure. The molecules can arrange to from bilayers with and the layers can arrange to stack on top of one another to form a lamellar sturucture. If the bilayers have a curvature to them they can enclose into a vesicle. The vesicle is a unique and interesting structure because a hydrophilic cavity is formed and enclosed by the bilayer. The supramolecular structure is dictated by molecular shape of the amphiphile and the hydrophobic/philic balance. Amphiphiles that have a linear shape are more likely to form Lamellar type packing and wedge shaped amphiphiles will from vesicles.

For Gene Delivery amphiphiles that from vesicles are of interest since the DNA can be included inside the cavity of the vesicle. The DNA would then be protected from degradation and should make it to the cell. Since the DNA is covered by lipids they can be referred to as Liposomes

 Initially anionic and neutral liposomes attracted interest because they were considered safe and compatible with bodily fluids and exhibited tissue specific gene transfer. However it was found that transduction to cell expression was low. Also DNA must be implemented into the vehicle to be used for gene delivery.
 * Anionic and Neutral Liposomes**

 Cationic liposomes form complexes readily with DNA. DNA is negatively charged because of the anionic phosphate ester backbone. The positively charged liposome can interact with the negatively charged cell membrane and therefore cell penetration can occur. The process by which lipoplexes deliver DNA to the nucleus is illustrated in Figure 2. The interaction between cells surfaces and lipoplexes are not yet known but studies have shown that proteoglycans play a key role. The liposome interacts with the cells surface and is taken up into the cell by endocytosis. The endosome formed from endocytosis is degraded and the DNA is released to the cytoplasm and then delivered to the nucleus. Since DNA is inactive when coated in the liposome one of the hypothesis is the interaction between the cationic lipolplexes and anionic cell lipids to neutralizes the complex and leads to the release of DNA.
 * Cationic Liposomes**

Membrane fusiogenic properties ( ability to fuse to cell) of lipoplexes play and important roel in cell uptake and endosomal escape of the DNA by destabilizing the anionic biological memebrane. 6



Introduction to Cyclodextrin
 * 

What is Cyclodextrin?**

Cyclodextrins (CDs)7,8 are rigid, cyclic compounds consisting of six, seven or eight glucose units linking by a- 1,4 glycosidic bonds for a, b, or g cyclodextrin respectively. The most commonly used CD is b and will be the CD seen in rest of the paper.



CDs are industrially produced from starch via cylcoglycosyltransferase enzymes and have the shape of a truncated cone with an inner cavity volume of 0.17- 0.42 cm3 and diameter of 0.49-0.79 nm. The CD cavities are hydrophobic and can form inclusion complexes with organic compounds in water or other hydrophilic solvents. Primary hydroxyl groups from the 6 position of the glucose units occupy the narrow mouth of the truncated cone forming the primary face, and secondary hydroxyl groups from both the 2 and 3 positions of the glucose units form the secondary face by occupying the other end of the cone.

Due the high number of reactive site on cyclodextrin, synthesis can be challenging. There are often problems of over and under substitution and selectivity. Since primary hydroxyl group are more reactive then secondary hydroxyl groups there is a significant differences in reactivity between the primary and secondary face of CD and chemistry can be applied to selectively modify one face of a CD.9,10,11,12
 * Challenges of CD Synthesis**

Synthetic strategies to selectively and efficiently modify the primary face of CD are widely at hand, however functionalization methodologies for the secondary face are scarce. The lower reactivity of the secondary hydroxyl requires protection of the primary hydroxyl groups then elaboration on the secondary face is carried out, followed by deprotection of the primary face and further functionalization.

The following Schemes outline some common synthetic strategies to achieve the selectivity and high yields.








 * PolyCationic Amphilic Cyclodextrin Derivatives for Non-Viral Gene Delivery

Why Cyclodextrin? ** There are many molecular scaffolds that are used in the formation of cationic liposomes. Many of which are polydisperse, poorly defined and difficult to characterized. A lot of these systems will not be clinically relevant due to complex regulatory requirement that must be overcome to obtain approval as a new drug candidate for human trials.

Macromolecules that are discrete, well characterizes that can bind, compact and deliver DNA in a non toxic and effective manner is a higher sought after structure. Cyclodextrin is attractive because it is a discrete well defined scaffold in comparison to other systems such as polymers. With the ability to chemically differentiate between the hydroxyl groups on the two faces CD can easily be synthesized into an amphiphilic molecule. CD are non-toxic and are commercially available for low cost and large quantities.

The general structure for Polycationic amphiphilic CDs is shown in the figure below. Hydrophobic alkyl groups are positioned on one rim of the CD and the cationic groups, which are protonated amines, are located on the other rim. Adjustment can be made to the structure by changing the linker and adjusting the length. The hydrophobic tail can be adjusted by changing the carbon chain length








 * Characterizing Ability of Amphiphilic Cyclodextrins for Non Viral Gene Delivery**

Once a derivative has been synthesized there are several experiments that must be preformed to assess its ability as a non-viral vector candidate. The requirements include

a) Ability of the derivative to compact DNA inside and around the vesicle b) Ability to encapsulate DNA c) Ability to protect DNA from Degradation so DNA can be delivered to cell d) Transfection efficiency


 * Experiments to Characterize CD/DNA complex Size/Shape Encapsulation ability**


 * a)** **Dynamic Light Scattering**
 * b)** **Transmission Electron Microscopy**
 * c)** **Gel Electrophoresis**


 * Protection from Degradation **

Deoxyribonucleases (DNases) are enzymes that catalyze the hydrolytic cleavage of the phosphodiester linkages in the DNA backbone. The CDplexes were exposed to DNase and assessed by Gel Electrophoresis and the relative protection of DNA by the complexes relative to naked DNA plasmid is reported.

The CD/DNA complexes are added to incubated cell cultures in well plates. Cells that have been used to study CD/DNA complexes are Chinese Hamster Ovary (CHO) cells, COS-7 (cell line) and BNLCL2 (Liver cells). The cells are incubated with the DNA-amphiphilic CD mixture for a period of time and then the transfection media is removed and the cells are allowed to grow for another period of time.
 * Transfection Efficiency**

The cells are lysed and analyzed using Flourescents and protein assay. A Luciferase encoding reporter gene can be used in the CD/DNA complex and a luminometer can be used to measure luciferase activity.


 * Cationic Amphiphilic Cyclodextrins Designed for Gene Delivery with Interesting Architectures and Synthesis**

The first cationic CD derivative to show the ability to form vesicles was published in 2002 by Darcy //et al.//13 The structure was designed to have the hydrophobic alkyl group on the primary side. In this synthesis the primary face was functionalized first and then the secondary face was functionalized with the polar ethylene glycol chain. The rationale for using his moiety is because the ethylene glycol chain is polar and therefore the hydrophobic head group is larger. The end of the ethylene glycol chain was functionalized to possess an amine that was protonated to achieve the cation.



Each step in this synthesis is reasonably yielding for cyclodextrin chemistry. The only draw back of this synthesis strategy is when introducing the ethylene glycol chains there is not control on the amount of ethylene carbonate that is added to each chain. This would lead to complex mixtures and challenging chromatography.

The self assembly of the cationic amphiphilic CD was investigated by Dynamic Light Scattering (DLS) and Transmission Electron Microscopy (TEM).

TEM Images of Vesicles

The dynamic size distribution for the non-ionic precursor (Second step of synthesis) was found to be 100-300nm. The electrostatic repulsion of the cationinc head group causes a larger membrane curvature and explains why the hexadecyl derivative has a smaller particle size and narrower distribution. The larger distribution and larger size of the hexyl derivative emphasizes the importance of the hydrophobic/hydrophilic balance of the amphiphiles has on the mode of aggregation. Since the polar head group with the cationic protonated amine and ethylene glycol chain are relatively large a longer hydrophobic alkyl tail is needed so the amphiphile is the right shape so the molecules can pack in a favourable manner to form well defined vesicles.

The group also investigated whether the vesicle could encapsulate guests. Fluorescent studies were carried out with Rhodieum B, a hydrophilic dye, to determine weather the dye was encapsulated inside the hydrophilic cavity

This first publication of a Polycationic amphiphilic CD derivative provides good evidence that there CD derivatives can form supramolecular structures that are capable of encapsulating guest. This derivative was not examined for biological purpose provides proof of principle that cationic Polycationic CD derivatives can be further explored as carriers of DNA for gene delivery.

This group also synthesized another series of cationic amphiphilic CDs.14 These derivatives were designed to have the Polycationic head group on the primary face of cyclodextrin and some derivatives had a biolabile ester group in the hydrophobic tail. The biolable ester in the hydrophobic tail may be a means of drug release from vesicles or nanoparticles.





Again no biological test or even physical experiments have been carried out but this is another example of the rationale behind synthesizing these non-viral vectors and offers a good protocol for synthesis of a library for investigation. Remember synthesis of CD derivatives is challenging and a scheme with high yielding reactions is a great success.


 * Polycationic Amphilipic CD derivative with Biological Results**

In 2009, Fernandez //et al// designed a series of compounds that displayed the cationic head group on the primary face of CD. 15 This series of cationic amphiphilic CDs contain a cysteamine segment. The group previously found that the ethylene spacer between the amine and CD core was improves the accessibility of the amine and there fore increases its reactivity.16 The enhanced accessibility of the amine should facilitate the formation of a salt bridge between the phosphate anions on the backbone of DNA. A variety of cationic head groups were introduced to the CD through Amine-isothiocyanate couplings. This linkage was chosen because it is a high yielding reaction and the link group has many hydrogen bonding sites. The belt of hydrogen bonding thiourea moieties are in the appropriate location to participate in cooperative binding to phosphate groups on the DNA skeleton. It is widely accepted that the charge compensation is not exclusively responsible for anion recognition in biological systems. The presence of a cooperative network of hydrogen bonding and electrostatic interactions is necessary. This reasoning is also true for phosphates.17

The head groups chosen will examine the structure activity relationship between the shape of the cationic hydrophilic head group and the transfection efficiency. Studies to examine the formation of CD/DNA complexes were positive**.**

To avoid the extra steps and therefore lower overall yield of the final compound the functionality on the primary face was established first, after which esterification of the secondary hydroxyl groups was carried out under basic conditions with the appropriate anhydride.
 * Synthesis **

Using the amine-isothiocynate coupling to link the CD core and the amine head group give two options for synthesis. The CD core could have the amine or the isothiocyante on the primary face. Both methods were explored and were equally as successful.

The R groups attached to the thiourea functional group were systematic variations in distance between the phosphate binding motifs (amine and thiourea functional groups) relative disposition and flexibility of linkers.


 * Structure Activity Relationship (SAR) Investigation of Linker Length and Flexibility**

In order for cooperatively of the non covalent intercalations of the phosphate binding motifs (H-bonding from thiourea functional group and electrostatic interactions from the cationic amine) to be proficient there must be an optimal length and flexibility. The length of the linker and the flexibility between thiourea and amine group was varied**.**

As the distance increased between the amine and thioureido functional groups increased the transfection efficiencies decreased. The longest alkyl linker with a C6 alkyl chain had a significantly lower transfection efficiency then ethylene and tetra-methylene linker.

The phenyl linkers proved to be more toxic and have lower transfection efficiencies then the CD derivatives with alkyl linkers.

The optimal linker length was the short and flexible ethylene group and this was used as a basis for further SAR studies.

The next structure modification was to increase the number of protonable nitrogens and therefore increase the density of cations on the amphiphile.
 * Increasing the Number of Protonable Nitrogens**



The linear R groups were found to have a lower transfection efficiency compared to the R group with the ethylene linker with only one protonable nitrogens. This is surprising because generally in biology analogs that display multiple binding motifs ( multi-valency) have higher binding and desirable results.

The next structural change was to modify the three dimensional structure of the cationic amines arms from a linear structure to a three dimensional dendritic structure

In this series it was found that the CD derivative with the ethylene linker and two cationic amine arms had almost the same transfection efficiency as the derivative with the ethylene linker with one cationic amine. Looking at the derivatives with the same amount of protonable amines it observed that the geometry of these derivative is more favorable then the linear geometry.

The CD derivative that displayed the highest transfection efficiency had two cationic amine arms on each primary site in the dendritic geometry and no ethylene spacer. This derivative had the optimal distance and geometry between the H-bonding network and the electrostatic interactions to have a favorable interaction with the DNA.

This paper presented an elegant and efficient synthesis of polycationic amphiphilic cyclodextrins designed for non viral gene delivery. Systematic structure activity relationship studies led to a derivative with the high transfection efficiency needed for gene therapy. This paper also underlines that precise nano-engineerig of these nanoparticles is possible and that changes at the atomic level can greatly affect the activity of these compounds. Click Clusters The same year that group also published in //Organic and Biomolecular Chemistry//.18 The rationale of design was similar to the derivative discussed previously. The synthesis of these compounds utilized the popular and high yielding “Click” reaction of Copper catalyzed coupling of an alkyne an azide to produce a triazole linkage.

Synthesis



The synthesis had few steps and was rather high yielding. After installing an azide group on the primary face of the CD the secondary hydroxyl groups were esterified to install the hydrophobic alkyl groups. A variety of alkynes were used for SAR studies.



A second library of derivative was synthesized to have a longer and more flexible linker group between the cationic amine groups and CD core**.**

The derivatives with the best transfection efficiencies had shorter hydrophobic alkyl groups on the secondary side. Generally the hexanoic derivatives offered better protection of the DNA and higher transfection efficiencies. This underlines the importance of having the right hydrophobic/philic balance.

The derivatives with the more flexible linker had a lower transfection efficiency then derivatives with the triazole linker directly attached to the 6-position of the glucose unit.


 * Assessment and Outlook for Poly cationic Amphiphilic Cyclodextrin Derivative as Non-Viral Vectors for Gene Delivery**

Due to safety concerns of viral vectors, success of gene therapy will likely be dependent on non-viral vectors. Although the chance of recombination of a defective viral vector into a replication competent virus is low, the consequences are drastic.

Cyclodextrin as a scaffold for non viral vectors has great potential but still faces some major challenges. CDs have the advantage of being monodisperse and discrete macromolecule that are well defined and can be completely characterized. This is an advantage over other polymeric systems which will face problems because they are poorly defined and characterized and will face problems due to complex regulatory requirements to obtain approval as new drug candidates for human trials. Also because of their well defined nature SAR studies can easily be carried out to optimize derivatives to their greatest potential as a non-viral vector. Already, CD derivatives have displayed promising results as a non viral vector**.**

Remaining challenges that I foresee for CD derivatives as non viral vectors are in the synthesis and selective targeting of cells.

Due to the multiple reactive sites on CD even high yielding reactions produce complex mixtures of under substituted and over substituted derivatives. Careful chromatography is needed in many steps of the synthesized derivatives, which would be costly and time consuming for commercial purposes. Also, some reaction conditions are incompatible for biological systems. For example, in the copper catalyzed alkyne-azide coupling, total removal of the copper catalyst is difficult to do and difficult to determine. Introducing copper into biological systems could have adverse affects, since copper is a common metal involved in biological processes.

The current CD derivatives do not have any ligands that can target specific cells. The papers suggest that the fusiogenic group is the hydrophobic alkyl groups that intergrate in the cells membranes. This strategy does not seem to have any specificity for particular cells and could introduce DNA to any cell.

Some future targets for CD derivatives as non viral vectors would be be include a ligand that would target desired cells for gene therapy**.**

**References**

1. Orkin SH: Molecular genetics and potential gene therapy. Clin Immunol Immunopathol, 1986; 40: 151 – 6

2. Rubanyi GM: The future of human gene therapy. Mol Aspects Med, 2001; 22: 113 – 42

3. Hauser H, Spitzer D, Verhoeyen E et al: New approaches towards // ex vivo //and // in vivo //gene therapy. Cells Tissues Organs, 2000; 167: 75 – 80

4. Chan L, Fujimiya M, Kojima H: // In vivo //gene therapy for diabetes mellitus. Trends Mol Med, 2003; 9: 430 – 35

5. Hacein-Bey-Abina, S. // et al. Science //** 302, ** 415–419 (2003).

6. J. Rejman, A. Bragonzi, M. Conese, Mol. Ther. 2005, 12, 468 – 474. 7//. Comprehensive Supramolecular Chemistry//; Atwood, J. L., Lehn, J.-M., Eds.; Vol. 3,

8. //Cyclodextrins//; Szejtli, J., Osa, T., Eds.; Pergamon: Oxford, U.K., 1996

9. //New Trends in Cyclodextrins and Derivatives//; Duchene, D.; Ed., Editions de Santé: Paris, 1991.

10. Croft, A. P.; Bartsch, R. A.; //Tetrahedron//, **1983**, 29, 1417-1474 and references therein.

11. D’Souza, V.; //Chem. Rev//., **1998**, 98, 1977-1996 and references therein.

12. Fügedi, P.; //Carbohydr. Res.//; **192,** 366-369//.// (b) Coleman, A. W.; Zhang, P.; Ling, C.-C.; Parrot-Lopez, H.; //Carbohydr. Res.//; **1992**, 224 307-309.

13. Baer, H. H.; Berenguel, A. V.; Shu, Y. Y.; Defaye, J.; Gadelle, A.;  González, F. S.; //Carbohydr. Res.,// **1992**, 228, 307-314.

14. Donohue, R., Mazzaglia, A., Ravoo, B.J., Darcy, R.; **2002**, “ Cationic // b // -Cyclodextrin Bilayer vesicles.” //Chem Comm//., 2864-2865. 15.Byrne,C., Sallas, F., Rai, D.K., Ogier, J., Darcy, R.; **2009** “Poly-6-cationic Amphiphilic Cyclodextrins Designed for Gene Delivery” //Organic & Biomolecular Chemistry.// 7: 3763-2771

16. Diaz-Moscoso, A., Le Gourriere, L., Gomez-Garcia, M., Benito, J.M., Balbuena, Ortega-Caballero, F., Guilloteau, N., Di Giorgio, C., Vierling, P., Defaye, J., Mellet, C.O., and Garcia Fernendez, J.M; **2009**, //Chem. Eu. J.// 15: 12871-12888 17.M. G_mez-Garc_a, J. M. Benito, D. Rodr_guez-Lucena, J.-X. Yu, K. Chmurski, C. Ortiz Mellet, R. Gueti_rrez Gallego, A. Maestre, J. Defaye, J. M. Garc_a Fern_ndez, J. Am. Chem. Soc. 2005, 127, 7970 - 7971.

18.P. A. Gale, S. E. Garcia-Garrido, J. Garric, Chem. Soc. Rev. 2008, 37, 151 –190; Mendex-Ardoy, A., Gomez-Garcia, Ortiz Mellet, C., Sevinallno, N., Giron, M.D., Salto, R., Gonzalez, S., Garcia Fernendez, J.M. **2009** “ Cylcodextrin ‘Click Clusters.’”//Organic & Biomolecular Chemistry.// 7: 2681-2684