Cellular delivery

Due to the predominantly hydrophilic nature of PNAs they do not readily cross lipid membranes [93] and enter living cells [94]. Therefore in order to explore the ex vivo and in vivo potential of PNA as an antisense and/or antigene reagent, a number of different transfection protocols have been devised over recent years. 

These protocols comprise a number of techniques involving either unmodified or modified PNA. 

Unmodified PNAs have been introduced to cells by microinjection [33], electroporation [60, 62, 76], co-transfection with partially hybridized DNA oligos [43, 61, 

direct dehvery using high concentrations of PNA [46], by the use of strep-tolycin-O permeabihzed cells [84] or by the employment of a mutant bacterial strain having a defective cell wall [64, 65]. 

To obtain an increased intrinsic capacity to transgress biological membranes, a number of different modifications have been introduced to PNA. These modifications include conjugation of PNA to Hpophilic moieties [51, 97, 98], conjugation of PNA to certain so-caUed ceU-penetrating peptides [49, 55, 56, 66, 99-102] and conjugation to different moieties, which are supposed to be internahzed by specific cellular receptors [48, 103-105]. The work on cellular dehvery of PNA is, like the related work on ex vivo and in vivo effects of PNA, very difficult to summarize conclusively. First of all, the pronounced diversity of the reporter systems employed makes it impossible to directly compare the studies. Secondly, the widespread use of fluorescence studies in spite of the many inherent pitfalls of this technique makes it sometimes difficult to judge even qualitatively whether a presented result actually indicates cellular uptake. We have recently published a comprehensive review on cellular dehvery of PNA [82], with a more detailed assessment of the PNA dehvery hterature. 

---

S., Hamer, )., Escobedo, J., Cohen, F., Radhakrishnan, R., Dwarki, V, and ZuCKERMANN, R.N. Lipitoids — novel cationic lipids for cellular delivery of plasmid DNA in vitro. Chem. Biol. 1998, 5, 345-354. 

Hamilton S. E., Simmons C.G., Kathir-iya I.S., Corey D.R. Cellular delivery of peptide nucleic acids and inhibition of human telomerase. Chem. Biol. 1999 6 343-351. 

Koppelhus U., Nielsen P.E. Cellular delivery of peptide nucleic acid (PNA). Adv. Drug Deliv. Rev. 2003 55 267-280... 

A. B. (2006) Inorganic nanopartides as carriers for efficient cellular delivery. Chemical Engineering Science, 61, 1027-1040. 

Hughes, M.D., Hussain, M., Nawaz, Q., Sayyed, P., and Akhtar, S. 2001. The cellular delivery of antisense oligonucleotides and ribozymes. Drug Discovery Today 6(6), 303-315. 

Brooks H, Lebleu B, Vives E. Tat peptide-mediated cellular delivery back to basics. Adv Drug Deliv Rev 2005 57(4) 559-577. 

A number of modified nucleotides have been tested and described in siRNA design. These are mostly modifications of the 2 OH group of the ribose. By the incorporation of chemically modified nucleotides into siRNAs, the on-target efficiency of the siRNAs can be increased (42 4). On the other hand, different types of siRNA off-target effects can be reduced by the use of chemically modified nucleotides immunostimulatory effects (reviewed in (20)) as well as sequence-dependent miRNA-like off-target effects (26, 44,45) and passenger strand incorporation (46, 47). In addition, chemical modifications can be used to improve the cellular delivery of siRNAs in living animals and are important tools to enhance the serum stability of siRNAs (48). 

H. Brooks, B. Lebleu, and E. Vives. Tat peptide—mediated cellular delivery Back to basics. Adv. Drug Deliv. Rev. 57 559-577, 2005. 

As an undergraduate, Jonathan Behrendt studied for a B.Sc. in Chemistry and Law from the University of Exeter, where he was awarded a 2.1 hons. degree in 2001. He went on to study for a Ph.D. in chemistry at University College London, with Dr. Helen Hailes. His Ph.D. research project focused on the use of polymeric supports for the synthesis of structurally defined oligomers and cyclic ethers. Having been awarded his Ph.D. in 2005, he is currently working on the synthesis and functionalization of polystyrene microspheres as cellular delivery vectors, in the research group of Dr. Andy Sutherland at Aston University. 

The rate of liposome accumulation in alveolar type-II cells is dependent on lipid composition. It is therefore possible to select liposome compositions displaying minimal interaction with these cells and thereby function as controlled-release systems for entrapped solutes. For example, liposomes composed of dipalmitoylphosphatidylcholine and cholesterol and containing entrapped sodium cromoglycate will provide sustained delivery of the drag for over 24 hours. Conversely other liposome compositions could be utilized for enhanced epithelial interaction and transport of the drug (e g. cationic lipids for the cellular delivery of the CFTR gene). 

Order, cellular delivery system or component Cell-specific homing sequence Organelle targeting sequence Signal sequence Cell-penetrating vector Delivery vehicle Order, immunological peptide Antigen... 

Key Words Cell-penetrating peptide noninvasive cellular delivery penetratin Tat transportan pVEC MAP protein transduction oligonucleotide transport plasmid delivery. 

Selection of an optimal CPP for delivering a specific cargo into a particular cell type, especially in vivo, is not necessarily a trivial task. Until now there are relatively few studies devoted to the comparison of the cellular delivery efficiency of different CPPs and the correlation to their side effects such as cytotoxicity and membrane destabilizing properties. Comparative data of the delivery efficiency of CPPs in vivo are even more scarce. 

Hydrogen bonds between specific pairs of nucleobases. A short PNA oligomer covalently coupled to a CPP has been used for the cellular delivery of complementary oligonucleotides (48) and plasmids (39). 

Biotin-avidin interaction. The cellular delivery capacity of several CPPs has been proven using complexes of biotinylated peptides with labeled avidin or strep-tavidin (23,27,37). Avidin could also serve as a scaffold for the assembly of molecules to be delivered into cells using biotinylated CPPs to facilitate the uptake process. 

Cellular Delivery of Peptide Nucleic Acid by Cell-Penetrating Peptides... 

PNA-NLS y Improved nuclear delivery y Improvement in cellular delivery is not fully validated (8)... 

---