rgd conjugated surfaces

8/3/2019 RGD Conjugated Surfaces

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RGD-Conjugated Surfaces in Biomaterials

Eric Falde, December 1, 2010

Overview

This review focuses on the synthesis, characterization, and applications of RGD-conjugated biomaterial surfaces. Particular focus is placed on liposomes and microbubble

applications for drug delivery and imaging, as these are emerging applications for the

technology.

Biomaterials are by definition foreign to the body, often possessing radically

different properties than the host system expects. This almost always leads to acute and

chronic inflammation then fibrous capsule growth, and far too commonly to material

degradation or rejection. Accordingly, there has been a great interest in developing

surfaces that mimic biological activity and function, not only to prolong product lifetime

but increasingly also to impart favorable biological interactions. The surface

immobilization of peptides and proteins, the sites of these interactions, has been the most

common approach for surface functionalization.

One of the best-studied surface peptide sequences is arginine-lysine-glycine (RGD),

which mimics the binding domain on the matrix protein fibronectin 1. This sequence has

been shown to bind to multiple integrins which are widely expressed, specifically 51,

V 1, 53, and 2b 3 2. Since the sequence was so short, it made surface functionalization

relatively simple, and was used early on to support cell growth and adhesion on polymer

surfaces 2,3 , but has seen much wider uses in recent years.

Besides providing structural stability, integrin attachment to RGD also begins

signaling cascades which promote cell survival 4. Loss of cell attachment sites induces

anoikis, a form of apoptosis, but conversely both the raf-Erk and JNK signaling pathways

are activated by integrin binding, and possibly the Jun-fos pathway as well. For example, an

RGD-bound 5 1 integrin induces the expression of Bcl-2, an anti-apoptotic agent, which

coaxes the cell to endure greater stresses 4. In fact, for at least some cell types, integrin

binding is necessary to progress from the G1 metabolic phase, and the types of bound

integrin affect differentiation 4.


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The force the cytoskeleton begins to apply on a bound integrin is enough to induce

endocytosis. When it was shown that RGD-conjugated liposomes and vesicles were

internalized, a new of route opened up for anything researchers wanted to bring into the

cell: drugs, tracers, nucleic acids, etc5. Especially the longer lasting, polyethylene glycol

(PEG)-shielded, stealth liposomes have proven very promising as delivery vectors when

functionalized with RGD 5,6 . Untargeted, these liposomes are very often used (and even FDA

approved) in cancer treatment applications where the extravasation effect, the added

permeability of tumor vasculature, is hoped to aid in delivery, though long circulation times

(>6 hours) and small size (


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Figure 1: Predicted secondary structure of the integrin attachment domain of fibronectin,

from Pierschbacher and Ruoslahti, 1984. The hydrophilic RGDS region is boxed, which was

shown to be necessary and sufficient for cell attachment 1

.

It is the boxed region of Figure 1 (or very similar regions found in fibrinogen and

vitronectin) that all RGD surface modifications seek to mimic. Note that the active site is

looped, and this conformation proved important in targeting specific integrins, as discussed

later. Integrins are structural adhesion proteins composed of two transmembrane

subunits, and , and smaller cytoplasm-side units which promote actin fibril formation

when bound integrins associate, as shown in Figure 2.

Figure 2: Diagram of integrin strucutre, binding, and clutering to form focal adhesions,from Giancotti and Rouslahti, 1999.


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The binding of cells to a rigid RGD-presenting surface consists of four overlapping

processes: cell attachment, cell spreading, organization of actin cytoskeleton, and

formation of focal adhesions 2. The actin fibrils which form in response to RGD binding

result in the structural support and endocytotic force, while association of additional

proteins to the bound complex results in various signaling cascades. Calveolin-1 is one such

protein and, through an unknown mechanism, associates with nearby integrins to promote

focal adhesion formation and endocytosis 4.

Though all are quite similar, some differences in expression and signaling the

between the RGD-specific integrins are relevant to surface modifications. The integrin 5 1

prefers to bind fibronectin, is widely expressed, and when bound activates Shc and

expression of Bcl-2 to enhance cell survival 4. The integrin V1 is very similar, but the

subunit has a higher affinity for the more cyclic RGD conformation of vitronectin7, and it is

highly expressed in the liver, but has no known survival signaling. Binding of the V 3

integrin, also partial to cyclic RGD, promotes survival of endothelial and melanoma cells

through p53 activation 4, and is highly expressed in endothelial cells during angiogenesis,

making it a promising target for cancer therapy.

Applications of RGD-Conjugated Surfaces

Early in-vitro cell culture relied on naturally produced, poorly characterized,

materials like collagen, gelatin or Matrigel. Using a chemically defined surface has obvious

advantages of greater control, specificity of interaction, and more reliable results.

Recently, interest in RGD surface modification has shifted to soft materials,

especially liposomes for drug delivery and diagnostics. In these applications, the surface is

instead a micelle or lipid bilayer that contains the drug (or nucleic acids) of choice, so the

synthesis is more involved and the reaction conditions are generally milder in order topreserve the vector and cargo. The simplest liposomes mix a phospholipid with an RGD-

conjugated (or primary amine-reactive) phospholipid, and then create liposomes using

sonication or reverse phase evaporation.


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Though the simple targeted liposomes did internalize well, they were cleared by

macrophages too quickly for practical in-vivo use. Therefore, the next added complexity

was to coat the liposomes in long chains of polyethylene glycol (PEG), creating targeted,

stealth liposomes, then conjugate RGD to the ends of the chains. Long PEG chains are very

hydrophilic, both increasing liposome solubility and serving as a steric cushion, greatly

slowing recombination of the liposomes (Ostwald ripening) and loss of liposome function.

This kind of liposome is especially attractive since it was FDA approved as a vector to

deliver high concentrations of the cancer drug doxorubicin, which has a circulation half life

of ~15 hours in order to take advantage of the extravasation effect 7. This approach seemed

too haphazard to some, who felt that the RGD group dangling at the end of a large PEG

chain was bound to cause non-specific interactions or induce degradation. Therefore, a

tiered architecture was developed, wherein the RGD group was attached to the end of a

shorter PEG group than those surrounding it.

Another application of RGD surface modification is in targeting contrast agents for

imaging. Targeted liposomes can also be created with MRI, or ultrasound imaging agents to

aid diagnosis or research. For example, superparamagnetic iron oxide nanoparticles

(SPIONs) have been conjugated to V 3-preferential RGD to locate carcinomas with MRI.

Similarly, microbubbles are essentially large liposomes (~5 m diameter), instead

containing gases such as perfluorobutane, and are used as ultrasound contrast agents

because the gas absorbs rather than reflects sound waves 14 . This technique has been FDA

approved in untargeted applications, but protein targeted microbubbles seem a logical and

well characterized improvement on the status quo. Targeting cell binding with RGD is

beneficial in this application to also target cancer cells to quantify and locate metastases,

though their size limits application to larger blood vessels and prevents extravasation. A

benefit of this technique, however, is that after imaging is concluded an ultrasound pulse

can burst the microbubbles in order to release a therapeutic in the affected area. Anotherinteresting quality of microbubbles is that if pulsed below the burst energy, the bubbles

swell but do not break. This fact has allowed the creation of a tiered architecture, in which

the RGD ligand is largely shielded from solution by conjugation to a shorter PEG chain, but


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then exposed when activated by a local ultrasound pulse 14 , greatly increasing targeting

control both temporally and spatially.

Chemistry of RGD Surface Modification

One of the strengths of RGD in functionalization is its small size and relatively

simple synthesis and reaction, as well as the large number of surfaces that can be modified.

Figure 3 some direct chemical conjugations which are reviewed.

Figure 3: RGD surface conjugation chemistry, from Hersel et al, 2003. (A) Thiol and

bromoacetyl-RGD, (B) Aldehyde and aminooxy-RGD, (C) Acrylate and thiol-RGD, (D)Maleinimide and thiol-RGD 2.

Briefly below are some of the methods used to attach RGD to various solid surfaces

for cell culture. In all cases, the linear peptide was synthesized separately then reacted at

the primary amine to form a covalent linkage. Glass was prepared for cell culture

passivating with a glycosyl-silane, then RGD-functionalized by activating with tresyl

chloride, which then attached the primary amine of RGD in sodium bicarbonate solution 8. A

poly(tetrafluoroethylene- co-hexafluoropropylene) surface was functionalized to cultureneurons by reducing the surface in sodium naphthalene in THF, followed by oxidation

using potassium chlorate or thallous ethoxide then tresyl chloride and then RGD 9. Acrylate

surfaces for controlling differentiation of embryonic stem cells have been RGD-conjugated

by addition of the tripeptide to a solution of acrylate-PEG- N-hydroxysuccinimide in a

sodium bicarbonate at room temperature, which was then blended with unmodified


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acrylates and photopolymerized 10 . Titanium was prepared for osseointegration by

alternately adsorbing and drying chitosan and hyaluronic acid, followed by reaction with

an RGD solution in the presence of EDAC and NHS in an MES buffer11 .

The simplest RGD-conjugated liposomes simply mix phospholipids with some RGD-

linked amphiphiles, create liposomes in a solution with the therapeutic, and wash. For

example, Nishiya and Sloan 12 used the amphiphiles diphosphatidylglycerol and dimyristoyl

phosphatidylcholine (DMPC) with some cholesterol (to increase fluidity) in a 1:6:3 ratio,

with 4 mol% also being N-3-(2-dithiopyridyl) propionyl phosphatidyl ethanolamine, all in

Hepes buffer. Then liposomes were formed in this solution using reverse phase

evaporation, in which the therapeutic solution was injected into the phospholipid mixture,

after which the pressure was lowered to evaporate the Hepes phase and concentrate the

lipids. Finally the liposomes were filtered to select for the desired size (between 1 and 0.2

m diameter) and incubated with GRGDSPC peptide, which attached to the thiopyridyl

groups at the primary amine 12 .

The synthesis of PEG-ylated liposomes is very similar, except one or more

phospholipid type is linked to a PEG group at the polar end. For example, Scheifflers et. al

used dipalmitoylphosphatidylcholine, distearoylphosphatidylethanolamine linked to 2000

g/mol PEG, and maleimide-PEG-2000 distearoylphosphatidylethanolamine dissolved in

cholorform/ethanol (2:1) in a ratio of 1.85:1.0:0.075:0.075, respectively. Then the pressurewas lowered to evaporate the solvents and forming the liposomes, which were filtered to

select for those under 50 nm. Finally, a cyclic RGDK peptide was synthesized with an

acetylthioacetyl group on the lysine, which was incubated with the peptides to react at the

exposed malemide ends as in Figure 3, (D). Microbubbles are synthesized in very much the

same manner, either by sonication or double reverse-phase evaporation, though the lipids

must remain more crystalline in order to trap the more mobile gas phase 14 .

Characterization of the Modification

Numerous methods have been developed to quantify the extent, location, and

effectiveness of RGD surface conjugation. The most important characterization methods

depend largely on the application, but can be broken down into acellular, cellular, and in-

vivo testing.


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The simplest acellular test for RGD conjugation on a surface is water contact angle;

hydrophobic surfaces will see decreased angles as the presence of zwitterionic RGD

increases. Depending on the initial surface, ATR-FTIR or XPS can indicate the molar ratio of

amines or carboxylate groups added 2. Alternatively, the amount of RGD added can be back-

calculated by measuring the concentration of unreacted peptide using HPLC 2. More

specifically, the degree of RGD surface conjugation can be more precisely quantitated by

conjugating with radiolabeled RGD (i.e. 125 I) and measuring by scintillation 9 or by tagging

RGD with a fluorescent protein11 . For liposomes, fluorescently labeled RGD-lipids provide

an easy marker and can even allow imaging of integrin clustering 15 . Though difficult and

expensive, integrin-conjugated surfaces can also be created which allow imaging and

quantitation of fluorescent RGD-liposomes 2.

Cellular tests are probably simpler for most labs to perform as cell adhesion to a

surface can be viewed under a light microscope. More quantitatively, spreading area and

confluency can be measured using confocal microscopy, AFM, or even SEM2. More in-depth

studies of signaling and differentiation induced by the RGD surface can be measured using

real-time PCR, and ECM deposition by immunostaining. Liposomal binding and cell

internalization rates can be measured using dyes and fluorescent microscopy or flow

cytometry. Organelle targeting is also important to ensure fragile cargo survives long

enough and is released in the cell properly to be effective, so confocal imaging and pH-sensitive tags such as RFP are very useful. Microbubble attachment can be imaged either by

ultrasound or light microscope. Crucial in these studies is the comparison to controls:

negative with non-functionalized surfaces, and missense with similar peptides such as

RGE2,3,5 . Without these controls and replicates of each condition, conclusions will be

tentative at best and very misleading at worst.


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Figure 4: Fluorescent microscope image of bound rhodamine-labeled PEG-RGD-liposomes,from Schiefflers et. al. Image taken intravitally from a mouse tumor 70 min after injection16 .

Finally, in-vivo tests are the ultimate standard by which RGD functionalization can

be measured, though of course this is only relevant so far as the entire product functions.

Mouse implants of functionalized surfaces and explant over time followed by histology is

the most direct way of measuring biocompatibility and induced cell growth. Again

comparison with controls is crucial, and often mice lacking immune systems are also tested

to control for inflammation and rejection effects. For liposomes, imaging using fluorescent

tags such as GFP or SPIONs is key to ensuring efficient targeting and delivery, as depicted in

Figure 4. Finally, improving the overall health of the animal (or person) is the ultimate goal,

which can be measured by a number of factors including body weight, tumor size, and

survival rates 16 .

Limitations and Improvements

Surface immobilization of RGD has been promising, but some limitations need to be

overcome. First, the added complexity and cost of functionalizing needs to be found worth

the benefits, which may not be the case in some applications.2 Second, the low specificity of

RGD for specific integrins, not to mention specific cell types is a major hurdle7. Finally,


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therapeutic load exit from liposomes once internalized is far from reliable; many vesicles

are transferred to lysosomes and drugs are degraded before they can be effective 7,13 .

However, there is significant progress being made on each of these issues. Though

costs have been slow to decline, basic science studies of integrin function and signaling give

a clearer picture of how RGD effects cells and when it might be beneficial. The use of cyclic

RGD has proved more effective in targeting 51 integrin, allowing more specific targeting

of cancer-specific cells 7. Further integrin studies indicated a second binding region in

fibronectin, PHSRN, which acts as a synergetic site with RGD17,18 . The length and

hydrophobic character of the linker between the two regions was studied in depth by

Kokkoli et. al, who developed a peptide amphihile which has been quite successful in

targeting liposomes and inducing internalization 7.

Summary

In conclusion, RGD-modification allows many types of cells to interact quite

favorably with a wide variety of surfaces and products, increasing cell adhesion, survival

signaling, and cell internalization. The abundance of RGD-binding integrins is something of

a weakness, however, lowering the cell specificity of targeting. For this reason, a wide array

of peptides are being surface immobilized, with promising results. For example, KGGRAKD

has ben used to target adipose tissue, YIGSR to target fibrosarcomas, VIP to target breast

tumors, and WIFPWIQL to target BiP proteins 7. However, due to its chemical simplicity,

widespread effectiveness and favorable signaling, RGD will likely remain as an

indispensible model for peptide immobilization for years to come.

References

1. Pierschbacher and Ruoslahti. Cell attachment activity of fibronectin can be

duplicated by small synthetic fragments of the molecule. Nature (1984) vol. 309pp. 30-33

2. Hersel et al. RGD modified polymers: biomaterials for stimulated cell adhesion

and beyond. Biomaterials (2003) vol. 24 pp. 4385-4415


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3. Brandley and Schnaar. Covalent attachment of an Arg-Gly-Asp sequece peptide

to derivatizable polyacrylamide surfaces: Support of fibroblast adhesion and

long-term growth. Analytical Biochemistry (1988) vol. 172 pp. 270-278

4. Giancotti and Ruoslathi. Integrin signaling. Science (1999) vol. 285 pp. 1028-

1032

5. Castel S, Pagan R, Mitjans F, Piulats J, Goodman S, Jonczyk A, Huber F, Vilaro S,

Reina M. RGD peptides and monoclonal antibodies, antagonists of avb3-

integrin, enter the cells by independent endocytic pathways. Lab Invest (2001)

vol 81 pp. 16151626.

6. Chiu et al. Electrostatic ligand coatings of nanoparticles enable ligand-specific

gene delivery to human primary cells. Nano Letters (2007) vol. 7 (4) pp. 874-

879

7. Pangburn et al. Peptide- and Aptamer-Functionalized Nanovectors for Targeted

Delivery of Therapeutics. Journal of Biomechanical Engineering (2009) vol. 131

pp. 1-20

8. Massia and Hubbel. An RGD Spacing of 440nm Is Sufficient for Integrin aV3-

mediated Fibroblast Spreading and 140nm for Focal Contact and Stress Fiber

Formation. Journal of Cell Biology (1991) vol. 114 (5) pp. 1089-1100

9. Tong and Shiochet. Peptide surface modification of poly (tetrafluoroethylene-co-hexafluoropropylene) enhances its interaction with central nervous system

neurons. Journal of Biomedical Materials (1998)

10. Ferreira et al. Bioactive hydrogel scaffolds for controllable vascular

differentiation of human embryonic stem cells. Biomaterials (2007) vol. 28 (17)

pp. 2706-17

11. Chua et al. Surface functionalization of titanium with hyaluronic acid/chitosan

polyelectrolyte multilayers and RGD for promoting osteoblast functions andinhibiting bacterial adhesion. Biomaterials (2008) vol. 29 pp. 1412-1421

12. Nishiya and Sloan. Interaction of RGD liposomes with platelets. Biochemical

and Biophysical Research Communications (1996) vol. 224 pp. 242-245

13. Schiffelers et al. Anti-tumor efficacy of tumor vasculature-targeted liposomal

doxorubicin. Journal of Controlled Release (2003) vol. 91 pp. 115-122


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14. Duncanson et al. Targeted binding of PEG-lipid modified polymer ultrasound

contrast agents with tiered surface architecture. Biotechnology and

Bioengineering (2010) vol. 106 (3) pp. 501-506

15. Marchi-Artzner et al. Adhesion of Arg-Gly-Asp (RGD) peptide vesicles onto an

integrin surface: visualization of the segregation of RGD ligands into the

adhesion plaques by fluorescence. Langmuir (2003) vol. 19, pp. 835-841

16. Schiffelers et al. Anti-tumor efficacy of tumor vasculature-targeted liposomal

doxorubicin. Journal of Controlled Release (2003) vol. 91 pp. 115-122

17. Feng and Mrksich. The Synergy Peptide PHSRN and the Adhesion Peptide RGD

Mediate Cell Adhesion through a Common Mechanism. Biochemistry (2004) vol.

43 pp. 15811-15821

18. Ochsenhirt et al. Effect of RGD secondary structure and the synergy site PHSRN

on cell adhesion, spreading and specific integrin engagement. Biomaterials

(2006) vol. 27 pp. 3863-3874

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