Nano Particle Drug Delivery

8/6/2019 Nano Particle Drug Delivery

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TARGETED NANOPARTICLE DRUG DELIVERYZ.B. Bilgiçer, H.-C. Chang, C. D’Souza-Schorey, B. Smith, and E. Y. Zhu

In the last decade, research in single-drug delivery by nanoparticles or nano-vesicleshas led to 24 commercial products with sales exceeding 5 billion US$. The next phase of research is on nanotechnology based directed drug delivery and controlled release. Using the

fundamental understanding of the biology of the diseased tissue, our goal is to designnanoparticles encapsulating multi-functional combinatoric drugs, and functionalize their surfaceswith recognition elements (such as RGD-peptidomimetics, antibodies, scFvs, etc.) that cantarget specific diseased cells. (1-7 )

Mammalian cells require oxygen and nutrients for their survival and are therefore locatedwithin 100 to 200 µm of blood vessels (the diffusion limit for oxygen). Multicellular organismsdevelop new blood vessels for growth and repair through a process called angiogenesis whichis regulated by a balance between pro- and antiangiogenic molecules. An imbalance in thisprocess can be initiated by numerous angiogenetic diseases such as malignant tumors,inflammatory, ischaemic, infectious and immune disorders.(8, 9)(10 ) Angiogenic diseases causethe blood vessels surrounding the tissue to grow larger and become more permeable (leaky)than healthy vessels by increasing the porosity of the vasculature. Liposomes and nano-

particles (with sizes between 100 nm to 200 nm) get trapped in these larger than averageporous regions of the blood vessels.(2, 11) This discovery has been used as means for passively targeting of angiogenic diseases with nanoparticles encapsulated drugs (Figure 1a),and the first applications reached the clinical trials in the mid-1980s. Passive targeting withnanoparticles, however, encounters multiple obstacles on the way to their target; these includemucosal barriers, non-specific uptake of the particle and non-specific delivery of the drug (as aresult of uncontrolled release).(1, 2 ) Therefore, two most important aspects of nanoparticle drugdelivery must be: i) the specific targeting of the diseased tissue with nanoparticles (appropriatesize and functionalization with antibodies—or other means of selective binding—providesmeans of enhanced delivery of drugs and reduced non-specific toxicity); and ii) the timedrelease of the drug (to prevent non-specific toxicity the drug must not diffuse out of the particlewhile it is still in the circulatory system, and must remain encapsulated until the particle binds to

the target).

Figure 1. a) Passive tissue targeting bynanoparticles in blood vessels. Particles gettFFped in the target tissue as a result of leaky vessels and ineffective lymphaticdrainage; b) active cellular targeting of nanoparticles with conjugated antibodies.

a) b)



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One way to overcome the first issue is to functionalize the nanoparticles with recognitionelements on their surfaces towards receptors present on the particular diseased tissue (Figure1b). The conjugated antibodies or short chain variable fragments (scFvs) will provide selectivebinding to the specific cell’s surface, and their endocytosis will be enhanced with suitablyadjusted binding affinities. We will refer to Başar Bilgiçer’s expertise in the physicalunderstanding of biomolecular interactions, antibody and scFv targeting, conjugation and

liposome chemistry to design the surface functionalized nanoparticles for active cellular targeting. The specificity can be fine-tuned by adjusting the surface density and the monovalentaffinity of the antibodies and/or scFvs for a particular disease. (12, 13)

To address the second issue, we will engineer multilayered nanoparticles, where eachlayer will contain one drug from the cocktail, and their release will be sequenced in accordancewith the appropriate timing of the delivery of each drug for combination therapy.Currently a significant amount of researchshows that combination therapy is moreeffective than traditional therapies.Furthermore, progressive and sequentialdelivery of multiple drugs from multi-layered

nanoparticles has been proven to be aneffective way of treating tumors. (14) Thekinetics of the release of the drug from thenanoparticles depend on: i) the strength of the interactions (usually the dominantinteraction is hydrophobic interactions)between the polymer and the drug as wellas the permeability of the polymer, when the drug is noncovalently encapsulated inside thenanoparticles; ii) the rate of hydrolysis of the covalent bond (usually an ester) between the drugand the polymer, when the drug is covalently conjugated to the polymer or the liposome. We willinvestigate the optimal chemistry for the polymer/drug combination for the desired timing of therelease of the drug from the nanoparticle.

There is already some preliminary effort in compound drug synthesis within the group.Elaine Zhu has successfully used AC electric field to synthesize multi-layered silicated largeliposomes and PEGylated polymeric nanoparticles. She has also stabilized the liposomes withmacro-counterions—nanocolloids of opposite charge from the ionic surfactants. Depending onthe field frequency, spherical liposomes of different dimensions and different number of lamellaelayers can be selectively synthesized (Figure 2). These multi-lamellae liposomes can beendowed with high mechanical stability when silicated or after condensation of oppositelycharged nanocolloid. The Russian-doll capsules allow encapsulation of different drugs withineach aqueous layer and perhaps hydrophobic drugs within the bilayers.

We also propose to develop a high-throughput microfluidic platform, with integratedimaging components, for screening nanoparticles (and possibly synthesize future drugcandidates) (Figure 3). We shall explore several new biocompatible methods for encapsulation

of different nanovesicles withdifferent drugs, a larger deliverycapsule that is mechanicallystable; and can yet release itsload, perhaps sequentially, upondocking. The PIs have experiencein synthesizing core-shell polymer colloids and multi-layeredliposomes. The microfluidic

Figure 2. Liposome nanoparticles produced by theZhu group.

Figure 3. Microfluidic platform for screening the size andloading of nanoparticles.



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platform will also provide means for selecting the nanoparticles that will havethe desired size and loading for providingmonodispersity to provide reliablereproducibility of the experiments.

Elaine Zhu is currently working

with Chia Chang to synthesize suchcompound capsules with microfluidicdevices such as the one shown in Figure3. The electrokinetic chip can impart theproperly tuned AC field on the liposomesat various stages on the chip to allowsequential encapsulation and wrapping athigh throughput.

Once the particles are synthesizedand selected, the biological assays thatwe will carry out to screen and studythese nanoparticles are described in the

schematics, and include the drug releaseassays as well as the binding and uptakestudies. Our ultimate goal is to show theefficacy of these targeted-controlledrelease nanoparticles in the appropriatepreclinical disease models. Morespecifically, we plan to use tumor xenograft or syngeneic mouse tumor models to study the efficacy of thesedrugs in inhibiting tumor growth,angiogenesis and metastasis. Furthermore, the pharmacokinetic studies will be accomplished inrats to asses the in vivo drug release rates from the nanoparticles. Finally bio-distribution

studies will be accomplished to determine the degree of specificity and selectivity in targeting of the diseased tissue by referring to the whole animal imaging expertise of Brad Smith’s group.The biodistribution and pharmacodynamic properties of the drugs that are released from theliposomes after reaching the target tumor is very different from that of free drug. It has beenshown that while free drug accumulates in all tissues because of its small size, liposomesaccumulate preferably in the tumors because of EPR effect. We plan to use free drug as anegative control and compare tissue distribution properties of free drug and liposomal drug. If the drug is released before it reaches the tumors, its biodistribution and pharmacodynamicprofile would resemble that of the free drug.

Further in vitro and in vivo experiments will identify whether the drug carryingnanoparticles function via either or both of the two pathways: i) after the particle is positioned inthe proximity of the diseased tissue, passive diffusion carries the drug from inside the particle to

inside the cells; ii) the particle gets endocytosed as a whole (or sometimes smaller endosomes),which is followed by the leaking of the drug inside the cell.

Endocytosis of larger endosomes or even the entire liposome delivery vehicle (> 10 nm)remains a mystery. (15 ) C. D’Souza-Schorey’s expertise in endocytosis of cells will allow us tofabricate microfluidic and imaging platforms for single cell assays. It may be quite possible thatendocytosis only occurs with some protein machinery rather than a physical phenomenon suchas rupture or fusion. After these preliminary studies, we shall develop a synthetic lipid bilayer platform or, if necessary, a single-cell platform for rapid drug screening.



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As bilayer rupture for such large nanocolloids requires an energy input in excess of 10 3 kT due to exposure of the hydrophobic surface, large nanocolloid internalization obviouslyinvolves a restructuring of the bilayer, such as protrusion, inversion or fusion. However, theexact nature of such topological rearrangement and how they are affected by dehydrationeffects of the endosome and the membrane receptor (or receptor aggregates) or an internalchange in the lipid composition due to complexification after docking remains unknown. It is

quite possible that the large endosome form complexes with the membrane proteins or with thebilayer in both mechanisms. Alternatively, there may be molecular transfer from the deliveryvehicle to the cell membrane. For example, if the nanocolloids Zhu uses to stabilize her vesiclesdissociate and condense onto charged protein sites on the membrane instead, the dockedvesicle would rupture spontaneously to release smaller endosomes or refuse with the cell toallow internalization of the entire vesicle. A large nanocolloid or a polyvalent counter-ion mayalso bridge the two surfaces, without dissociation from one, to produce a complex that favorsfusion.

It is quite possible that all the endocytosis routes can occur and specific drug designrequires careful screening of the pertinent internalization mechanism. (15 ) Such hypotheses oncolloid/liposome interaction will be first examined with synthetic lipid bilayers with the AFM,FCS, TIRF and Surface Force Apparatus in Zhu’s lab. We shall support the lipid bilayers on

nanopillar supports fabricated to allow the study of internalization before testing the platformagainst real cell membranes. Release of specific chemicals from nanofluidic channels will beused to examine the effects of certain stimuli. Obviously, a synthetic bilayer platform would beeasier to run than one with real cells. The science of incorporating membrane proteins into theartificial bilayers while retaining their bioactivity is well-developed and we shall include specificreceptors for our antibodies.

Ultimately our studies will establish the most efficient nanoparticles system with the rightqualifications, and the chosen system will be carried into clinical trials with the intention of eventually improving patient outcome. Meanwhile, in the process, we will also be addressingseveral unknown biophysical issues: how an antibody functionalized nanoparticle interacts witha lipid bilayer membrane, how endosomes released by the vehicle are internalized viaendocytosis, how membrane protein aggregate patterns change due to docking with antibodies

functionalized to delivery vehicles and whether the protein aggregation can contribute to thedrug release and internalization.

The Faculty Fellow (FF) we seek is someone with extensive research experience innanoparticle drug delivery field who can choose and design the critical biological study plan toevaluate and select the targeted drug-bearing nanoparticles with the highest physiologicalrelevance. The FF will complement the liposome and polymeric drug (antibody) synthesisknowledge in Bilgicer’s group, imaging expertise in Zhu’s and Smith’s labs, the biologicalstrength of D’Souza-Schorey’s group and the microfluidic/nanofluidic fabrication know-how inChang/Bohn’s labs. The FF will have responsibilities of a project leader and project manager,and will be expected to reach out to set up the right collaborations to make progress in allaspects of this project to bridge biology with engineering. As such drug synthesis and fabricationplatforms should be of interest to the pharmaceutical industry and can lead to new start-ups,

some one with start-up experience would also be desirable.

References:

(1) Alonso, M. J. (2004) Nanomedicines for overcoming biological barriers. Biomedicine &

PharmacotheFFy 58, 168-172.(2) Couvreur, P., and Vauthier, C. (2006) Nanotechnology: Intelligent design to treat

complex disease. Pharmaceutical Research 23, 1417-1450.



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(3) Duncan, R. (2006) Polymer conjugates as anticancer nanomedicines. Nature ReviewsCancer 6 , 688-701.

(4) Duncan, R., Vicent, M. J., Greco, F., and Nicholson, R. I. (2005) Polymer-drugconjugates: towards a novel approach for the treatment of endrocine-related cancer.

Endocrine-Related Cancer 12, S189-S199.

(5) Farokhzad, O. C., Cheng, J. J., Teply, B. A., Sherifi, I., Jon, S., Kantoff, P. W., Richie, J.P., and Langer, R. (2006) Targeted nanoparticle-aptamer bioconjugates for cancer chemotheFFy in vivo. Proceedings of the National Academy of Sciences of the United

States of America 103, 6315-6320.(6) Farokhzad, O. C., and Langer, R. (2006) Nanomedicine: Developing smarter theFFeutic

and diagnostic modalities. Advanced Drug Delivery Reviews 58, 1456-1459.(7) Ferrari, M. (2005) Cancer nanotechnology: Opportunities and challenges. Nature Reviews

Cancer 5, 161-171.(8) Carmeliet, P., and Jain, R. K. (2000) Angiogenesis in cancer and other diseases. Nature

407 , 249-257.(9) Carmeliet, P. (2005) Angiogenesis in life, disease and medicine. Nature 438, 932-936.

(10) Cancers (e.g. solid tumors), autoimmune diseases (such as rheumatoid arthritis andinflammatory bowel disease) and cardiovascular diseases (e.g. atherosclerosis) are some

examples.(11) Guo, R., Zhang, L. Y., Jiang, Z. P., Cao, Y., Ding, Y., and Jiang, X. Q. (2007) Synthesis

of alginic acid-poly[2-(diethylamino)ethyl methacrylate] monodispersed nanoparticles bya polymer-monomer pair reaction system. Biomacromolecules 8, 843-850.

(12) Park, J. W., Hong, K. L., Kirpotin, D. B., Colbern, G., Shalaby, R., Baselga, J., Shao, Y., Nielsen, U. B., Marks, J. D., Moore, D., Papahadjopoulos, D., and Benz, C. C. (2002)

Anti-HER2 immunoliposomes: Enhanced efficacy attributable to targeted delivery.Clinical Cancer Research 8, 1172-1181.

(13) de Menezes, D. E. L., Pilarski, L. M., and Allen, T. M. (1998) In vitro and in vivotargeting of immunoliposomal doxorubicin to human B-cell lymphoma. Cancer Research

58, 3320-3330.(14) Sengupta, S., Eavarone, D., Capila, I., Zhao, G. L., Watson, N., Kiziltepe, T., and

Sasisekharan, R. (2005) Temporal targeting of tumour cells and neovasculature with ananoscale delivery system. Nature 436 , 568-572.

(15) Chernomordik, L. V., and Kozlov, M. M. (2003) Protein-lipid interplay in fusion andfission of biological membranes. Annual Review of Biochemistry 72, 175-207.

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Nano Particle Drug Delivery