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Goux CHEM 4390 Daniel Gonzalez

Daniel Gonzalez CHEM4390 GOUX 2016

Antimicrobial Peptides Conjugated to PAMAM

Dendrimers for Anti-Cancer Drug Delivery Platforms: A Brief

Literature Review

The University of Texas at Dallas

Dr. Warren Goux: CHEM 4390

Daniel Gonzalez

Image: PAMAM Dendrimer, Diallo, Mamadou S. 2006



Researchers have been increasingly investigating cytotoxic peptides, that is, an amino

acid sequences known to cause cellular death. There are many well-known non peptide cytotoxic

compounds that are incredibly effective toxins to either bacteria or eukaryotic cells such as

penicillin, cyanide, cis-platin ect. Antimicrobial peptides however may hold novel medicinal

properties, due to the variable conformation, charge and sequence modification of the peptides

themselves. These factors contribute to incredible amounts of cell/receptor selectivity, which in

turn, can mean specific therapies for different cancers or bacterial infections.

Peptides are interesting for the many different mechanisms by which they can induce

cytotoxic effects. In the case of Alzheimer’s disease it has been shown by Zhao et al., that a

simple six amino acid residue specific to the microtubule binding repeat of Tau protein, known

as T-peptide, is capable of killing 80% of the cultured mouse hippocampal neurons. T-peptide

attributes it’s cytotoxic capabilities to a mechanism that involves forming protein aggregates

inside the cell resulting in neuro fibrillary tangles, which a hallmark of an Alzheimer’s diseased

brain tissue. The exact mechanism as to how these tangles cause cell death is not fully

understood, yet despite this, researchers at the University of Texas at Dallas working with Zhao,

demonstrated by testing multiple cell strains, that kidney cells showed a much higher resilience

to t-peptide. This finding suggests intuitively that certain cell types are specifically susceptible to

peptide induced modes of cytotoxicity.

Modes of cytotoxicity however, are not necessarily limited to the physical uptake of toxic

peptides into the cytoplasm of the target cell. Naturally occurring antimicrobial peptides such as

Megainin, Protegrin, and Melittin have all been demonstrated to cause cell death by creating

pores in the cytoplasmic membrane (Ludke, 1996). In the case of Megainin, which occurs

naturally on the skin of Xenopus laevis, the African Clawed Fog, the peptide consists of a 23



residue amphiplic alpha- helix that can integrate longitudinally into bacterial cell membranes. As

demonstrated by Ludtke at Rice University, when Megainin associates with 6 other membrane

bound Megainin peptides, they can coordinate to form a toroidal pore in which the lipid

membrane folds back on itself with the Megainin peptides imbedded in the membrane. These

pores are then responsible for the death of the bacterium. Protegrin, and Melittin have similar

toroidal -pore forming mechanisms as well.

Alamethicin on the other hand, forms cellular membrane pores, but does so by forming

beta-barrels that orient themselves perpendicularly in the cytoplasmic membrane. These beta-

barrels associate to form a “Barrel-Stave” in which a channel is created by a hydrophilic pore

formed between multiple domains of Alamethicin.

Figure. 1

Figure 1. The Barrel Stiev Model vs Toroidal Pore model of membrane cytotoxicity in antimicrobial peptides

The formation of pores to kill cells is not new in literature. With this in mind

Sinthuvanich et al. in 2012 engineered a beta-hair pin peptide designed to specifically target

cancer cells. Sinthuvanich’s approach entailed creating a 18-residue peptide SVS-1 that



remained unfolded in solution, but when in proximity of a negatively charged membrane would

fold with a beta-hair pin loop allowing the hydrophobic portions to be permeable in the lipid

membrane. When these peptides associated with one another in the membrane, they could form

pores by mechanisms similar to that of Alamethicin, causing cell death.

Figure 2.

Figure 2. Sinthuvanuch’s SVS-1 peptide mode of action

Clearly, for mechanisms involving perforating the cellular membrane, charge, polarity,

and pore forming mechanism seem to be obvious variables to be taken into consideration. In the

case of Sinthuvanich’s et al., researchers took advantage of the negative charged cellular surface

of cancer cells and utilized the cationic affinity of a positively charged peptide.



Normal mammalian cells have phosphatidylcholine and sphingomyelin distributed

throughout the outer leaflet of the lipid bilayer. The inner leaflet contains higher concentrations

of phosphatidylethanolamine and phosphatidylserine, which give rise to a net negative charge. In

cancerous cells, it has been observed that phosphatidylserine is transferred across to the outer

leaflet by aminophospholipid translocases and scramblases, resulting in a negative charge (Riedl

et al 2011). This negative charge was the key to SVS-1’s ability to selectively target cancer cells.

Figure 3

Figure 3. Normal Distribution of phosphatidylserine in mammalian phospholipid bilayer.

Interesting prospects to consider then, are what other peptides could be used for anti-cancer

purposes, and how could current anti-cancer peptides be made more potent with a higher degree

of specificity?

Dendrimers, large star shaped polymers, are becoming more prevalent as possible drug

delivery systems, specifically in the case of antimicrobial, or targeted peptide delivery.

Polyamidoamine, (PAMAM) dendrimers, are simple hyper branched dendrimers synthesized by

Michael addition, followed by alternating amidation reactions between methylacrilate and

diethylamine.



Figure 4. Synthesis Scheme of PAMAM Dendrimer

Researchers at the University of Michigan Ann Arbor conjugated RGD-4C, a peptide that

specifically bonds to a the αVβ3 marker necessary for angiogenesis, to a PAMAM dendrimer, and

observed increased selectivity as well as a longer diffusion rate of the peptide away from the

target cells (Shukla et al,2005). According to Shukla, the author of the study, the dendrimer

scaffold slowed dissociation by approximately 522 times as compared to the free peptide

(Shukla et al, 2005). Additionally, Chinese researchers Liang Han, and Rongqin Huang in 2010,

successfully conjugated a heptametric peptide termed T7 that specifically targets transferrin

receptors and observed increased cellular uptake of the PAMAM dendrimer. These result suggest

that multiple peptide conjugation on a single dendrimer exerts a synergistic effect on binding

efficiency, and this effect should be observed in other small peptides bound to the PAMAM as

well.



Figure 5. How Dendrimers incorporate into Cell Membranes

Image: Srinivas Parimi et all, 2010

Like antimicrobial peptides, dendrimers’ aliphatic chains allow them to readily diffuse

through cellular membranes. It has been observed that more cationic dendrimers tend to diffuse

more readily into the cellular membrane. Higher generation dendrimers of PAMAM have

enhanced interaction with dimyristoyl-sn-glycero-3-phosphotidylchoine (DMPC) liposomes

(Ottavian 1998) and high Levels of dendrimer internalization are also associated with

cytotoxicity, as demonstrated with arginine- and ornithine conjugated PAMAM dendrimers in

Caco-2 cells (Klainert 2005). All of these results suggest the following trends: larger, more

cationic dendrimers, are most cytotoxic due to their increased ability to permeate the

membrane.



One might be inclined to believe that if more positive charge was attached to a scaffold such as

a PAMAM dendrimer, the cationic effect observed by Sinthuvanich’s SVS-1 peptide would be

magnified. This has been confirmed by multiple studies such as those performed by Shukla and

others. Despite these gains of higher selectivity, it has been demonstrated that PAMAM

dendrimers are shown to cause concentration/ generation dependent cytotoxicity and hemolysis.

In an article in 2003, Jevprasesphant et al., investigated the cytotoxicity of PAMAM dendrimers

using Caco-2 cells and concluded that anionic or ,half generation dendrimers, show significantly

low toxicity in comparison to their respective cationic family (Madaan et al 2014). According to

the very first in vivo toxicological study, doses of 10 mg/kg appeared to be non-toxic up to fifth

generation of PAMAM dendrimers, when G3, G5 and G7 generations were injected into mice

and all the tested generations were found to be non-immunogenic. Using flow cytometry and

microscopic analysis it was demonstrated that cationic fluorescein isothiocyanate labeled G7

PAMAM dendrimers caused platelet disruption, whereas neutral (hydroxyl terminated) and

anionic (carboxyl terminated) PAMAM dendrimers did not alter platelet morphology or their

function (Madaan et al 2014).

Clearly, adding more positive charge to a dendrimer scaffold to increase cancer and

apoptotic selectivity is not a feasible option without dramatically increasing the overall toxicity

of the dendrimer itself. With this in mind though, it would be interesting to study the cytotoxic

effect of a .5 -1.0 generation PAMAM dendrimer with only 8 and 16 branching terminals

respectively, due to the fact that they are significantly smaller than their generation 4 and up

derivatives. Despite this, to dismiss the usefulness of a PAMAM dendrimer scaffold solely on

these cytotoxic effects would be premature. PAMAM dendrimers can be terminated with



carboxyl groups rather than amines, giving the molecule a more anionic nature and reducing the

cytotoxicity while maintaining solubility characteristics and useful functional moieties. Despite

cytotoxicity issues associated with higher generation PAMAM Dendrimers, surface modification

by adding poly ethylene glycol has been demonstrated to result in high cell viability.

Figure 6. Cell Viability of PAMAM Dendrimer Dox and Poly Ethylene Glycol

(Ling Han 2010)



Lower generation acetylation of PAMAM was similar in cytotoxicity to high generation poly

ethylene glycol substitutions. However, the attachment of large aliphatic groups to PAMAM

dramatically reduced the cytotoxicity ,but at the same time, increased molecular hydrophobicity.

This increase in hydrophobicity has been demonstrated to cause dendrimers to aggregate in

aqueous solutions (Jevprasesohant, 2003).

Furthermore, due to the branched nature of the dendrimer, different functional peptides or

drugs could be attached for unique pharmakinetic roles. For the role of specificity and targeting

apoptotic cells, phosphatidylserine specific peptides could be functionalized onto the dendrimer,

reducing the cationic charge but maintaining selectivity. Annexin V ,C2A domain of

Synaptotagmin, Lactadherin, and the peptide sequences:

LIKKPF, TLVSSL, CLSYYPSY, SVSVGMKPSPRP, FNFRLKAGAKIRFG,

have all been demonstrated as phosphatidylserine targeting peptides (Smith, 2012). A second

research group independently identified CLSYYPSY as a novel PS-interacting octapeptide as

well. A fluorescein labeled version of this peptide specifically targeted to tumor vasculature and

apoptotic tumor cells in xenografts treated with camptotheicn (smith 2012).



(Istev’an, 2005)

Other researchers successfully bound Taxol to PAMAM dendrimers for drug delivery at a

functionally active OH group on terminal carboxylic acids using a generation 5 dendrimer

scaffold. The researchers partially acetylate the G5 dendrimer to maintain solubility and

subsequently measured cytotoxic effects in concentrations as low as 100nM. It was demonstrated

that the dendrimer conjugated with Taxol was more cytotoxic than the Taxol or the dendrimer

alone (Istev’an, 2005).

With so many different practical applications for PAMAM dendrimers, and the wide

variety of already known, or currently being researched cancerous cell signals, it seems as

though one could be able to synthesize a plethora of drug delivery systems similar to the T7-

Dox-Peg-PAMAM system synthesized by Han et al. Choosing a low generation PAMAM

dendrimer, or attaching functional polyethylene glycol chains to a larger generation dendrimer,

has been demonstrated to create generally non-toxic scaffold systems. When these said systems

are in place, the coordination of antimicrobial peptides, targeting peptides, and cytotoxic drugs

have been demonstrated to create specific cancerous cell targeting therapeutic drug delivery

systems.



Work Cited:

Bryan A. Smith and Bradley D. Smith, Biomarkers and Molecular Probes for Cell Death Imaging and Targeted Therapeutics: Bioconjugate Chem. 2012, 23, 1989−2006

Chomdao Sinthuvanich, et al Anticancer β-Hairpin Peptides: Membrane-Induced Folding

Triggers Activity J. Am. Chem. Soc. 2012, 134, 6210−6217

Fischer D, Li Y, Ahlemeyer B, Krieglstein J, Kissel:In vitro cytotoxicity testing of polycations: influence of polymer structure on cell viability and hemolysis. TBiomaterials. 2003 Mar; 24(7):1121-3

Istva’n J. Majoros, Andrzej Myc, Thommey Thomas, Chandan B. Mehta, and James R. Baker, Jr. Received August 25,2005, Revised Manuscript Received Decembber 2, 2005 Biomacromolecules 2006, 7, 575-579 Jevprasesohant, R., Penny, Jalal, R., Attwood, D., McKeown, N. B., and D’Emanuele, A. (2003) The Influence of surface modification on the cytotoxicity of PAMAM dendrimers. Int. J. Pharm. 252, 263-266

Kanika Madaan, Sandeep Kumar, Neelam Poonia, Viney Lather,1 and Deepti Pandita Dendrimers in drug delivery and targeting: Drug-dendrimer interactions and toxicity issues J Pharm Bioallied Sci. 2014 Jul-Sep; 6(3): 139–150. doi: 10.4103/0975-7406.130965

Kelly Zhao, et al Neuron-Selective Toxicity of Tau Peptide in a Cell Culture Model of Neurodegenerative Tauopathy: Essential Role for Aggregation in Neurotoxicity Journal of Neuroscience Research 88:3399–3413 (2010)

Klajnert, B.; Epand, R.M Int J. Pharm. 2005, 305, 154-166.

Liang Han, Rongqin Huang, Shuhuan Liu, Shixian Huang, and Chen Jiang Received May 31,2010; revised August 20, 2010; accepted September 21, 2010 Molecular Pharmaceutics vol. 7, no. 6, 2156-2165 Ottaviani M.F.; Matteini, P.; Brustolon, M; Turro, N. J.; Jockusch, S.; Tomalia, D.A. J. Phys. Chem. B. 1998, 102, 6029-6039. Rameshwer Shukla , Thommey P. Thomas , Jennifer Peters , Alina Kotlyar , Andrzej Myc and James R. Baker, Jr. Tumor angiogenic vasculature targeting with PAMAM dendrimer–RGD conjugates 2005 Rohit . Kolhatkar, Kelly M. Kitchens, Peter W. Swaan, Hamidreza Ghandehari, Surface Acetylation of Polyamidoamine (PAMAM_ Dendrimers Decreases Cytotoxicity while Maintianing Membbrane Permeability: Received December. 18, 2006; Revised Manuscript Received Septembber 13, 2007 Bioconjugate Chem. 2007, 18, 2054-2060



Sabrina Riedl, Beate Rinner, Martin Asslaber, Helmut Schaider, Sonja Walzer,Alexandra Novak , Karl Lohner, Dagmar Zweytick, In search of a novel target — Phosphatidylserine exposed by non-apoptotic tumor cells and metastases of malignancies with poor treatment efficacy Biochimica et Biophysica Acta 1808 (2011) 2638–2645

Steve J. Ludtke Biochemistry 1996,35, 13723-13728 Membrane Pores Induced by Magainin

Steve Ludtke Biochemistry 1995,34, 16764-16769 Membrane Thinning Caused by Magainin 2 Figure 3 https://www.nap.edu/read/21733/chapter/5#26

Yoonkyung Kim, Athena M. Klutz, and Kenneth A. Jacobson: Systematic Investigation of Polyaminoamine Dendrimers Surface-Modified with Poly(ethylene glycol) for Drug Delivery Applications: Synthesis, Characterization, and Evaluation of Cytotoxicity Bioconjugate Chem. 2008, 19, 1660-1672

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