Drug Delivery in Cancer

Amit SoniInt. PhD NCU

S0626

Diagnostics

Therapeutic mechanisms

Targeting

Nanoparticle

Antigen labelling

Extracellular biomarkerdetection

Bioanalytesensor

Gene therapy

Endosomal escape(protein, small moleculeor siRNA)

MitochondrialDNA

Induction ofautophagy

Antibody

Selective endosomaltra c ing

AptamerPeptide

Small-molecule ligand

Lipoplex

Charged membranes

Extracellular drug release

Cell captureand removal

Immune cellrecruitment

Hyperthermia

Cell backpack

_

_

Course Supervisor : Dr. Jayanta Haldar, NCU

What is Tumor??Tumors are a basically sign of inflammation. It is a fluid-filled lesion that may or may not be formed by an abnormal growth of neoplastic cells that appears enlarged in size.

Benign:

1) Slow growing2) Capsulated

3) Non- invasive

4) Do not metastasize

Malignant:

1) Fast growing2) Non-capsulated3) Invasive & infiltrate

4)Metastasize

What is cancer?

1.The word cancer is derived from crab because cancers like a crab, they "grab on and don't let go." 2.The term cancer specifically refers to a new growth which has the ability to invade surrounding tissues, metastasise and which may eventually lead to the death.

A dividing lung cancer cell. Credit: National Institutes of Health

Cancer harms the body destroying healthy tissue in a process called invasion.

Cell manages to divide and grow, making new blood vessels to feed itself in a process called angiogenesis.

HOW TUMOR BECOMES CANCER??

Nature Reviews | Cancer

ab

c

de f

g

Cancer cell

Endothelial cell

Epithelial cell

Stromal cell

Basement membrane

Primary tumour

Secondary site

PlateletImmune cell

Shear ratesThe velocity gradient that is the relative velocity at which one layer of the fluid flows over an adjacent layer of the fluid.

Kupffer cellsA type of macrophage that lines the sinusoid walls of the liver and that removes toxins present in blood coming from the digestive tract. Involved in the breakdown and recycling of red blood cells and haemoglobin.

siRNA-loaded lipid nanoplexes to liver hepatocytes62,65,66. Conjugating monoclonal antibodies that target BBB receptors to the surface of nanoparticles has also been reported to increase uptake into the brain parenchyma67.

Nanoparticles have been shown to be trafficked to the brain after being taken up by cells in the circulation. For example, sugar-coated nanoparticles can be phago-cytosed by leukocytes and macrophages55,68. Such cells accumulate at sites of BBB degradation that are associ-ated with disease, and they have the capacity to infiltrate the brain69–71. By targeting these cells in the circulation, nanoparticles might be trafficked into the brain, where they can ultimately be released68,72. The ability of cells to infiltrate deep tissue, cross biological membranes and target disease sites has made them attractive carriers of nanoparticles73,74.

Lymph nodes, which are linked by lymphatic ves-sels, are distributed throughout the body and have an

integral role in the immune response. Dissemination of cancer cells through the lymph network is thought to be an important route for metastatic spread. Tumour proximal lymph nodes are often the first site of metasta-ses, and the presence of lymph node metastases signifies further metastatic spread and poor patient survival75. As such, lymph nodes have been targeted using cell-based nanotechnologies.

Certain characteristics are associated with prefer-ential (but not exclusive) nanoparticle trafficking to lymph nodes following intravenous administration76–79. Targeting is often an indirect process, as receptors on the surface of leukocytes bind nanoparticles and trans-fer them to lymph nodes as part of a normal immune response76. Several strategies have been used to enhance nanoparticle uptake by leukocytes in circulation. Coating iron-oxide nanoparticles with carbohydrates, such as dextran, results in the increased accumulation of these nanoparticles in lymph nodes78–80. Conjugating peptides and antibodies, such as immunoglobulin G (IgG), to the particle surface also increases their accumu-lation in the lymphatic network81. In general, negatively charged particles are taken up at faster rates than posi-tively charged or uncharged particles76,77. Conversely, ‘stealth’ polymers, such as polyethylene glycol (PEG), on the surface of nanoparticles, can inhibit uptake by leukocytes82–84, thereby reducing accumulation in the lymph nodes.

Lymph node targeting may be achieved by other routes of administration. Tsuda and co-workers85 reported that non-cationic particles with a size range of 6–34 nm, when introduced to the lungs (intrapulmo-nary administration), are trafficked rapidly (<1 hour) to local lymph nodes. Administering particles <80 nm in size subcutaneously also results in trafficking to lymph nodes86,87. Interestingly, some studies have indicated that non-pegylated particles exhibit enhanced accu-mulation in the lymphatics and that pegylated particles tend to appear in the circulation several hours after administration86.

The liver is a frequent site of metastasis. This well-vascularized tissue, with low shear rates and accessible capillaries, provides a ‘friendly soil’ for cancer cells. Many researchers think that targeting the liver is a simple task because various injected agents can accumulate in this organ. However, recent studies that aimed to deliver siRNA to liver hepatocytes showed that precise particle engineering is required to pass through the fenestrae that are present in the liver endothelium88. In general, intravenously administered nanoparticles accumulate in activated Kupffer cells that reside within and near the liver vasculature and so do not reach the hepatocytes89. Active targeting through endothelial fenestrations to hepatocytes has been facilitated via ApoE adherence to the particle surface while in the circulation, or by con-jugating carbo hydrates, such as N-acetylgalactosamine (GalNAc), to the surface of the particle90. This is an example of how organ and cell targeting are often interconnected. An ongoing clinical trial is using lipid nanoparticles as siRNA carriers for treating liver cancer and metastases91.

Figure 1 | The steps of metastasis and opportunities for therapeutic intervention. Metastasis requires several steps, each of which presents an opportunity for new therapies. First, metastatic cells must break free from the primary tumour. To accomplish this, cancer cells (a) reduce adhesion to neighbouring cells and (b) clear a path for migration into the vasculature-rich stroma210–213. Once at the vasculature, cells can freely enter the bloodstream if the vasculature is discontinuous, such as in certain regions of the liver, bone marrow and kidneys. Intravasation (c) is required if the vasculature is continuous; cells either cause endothelial cell retraction by releasing compounds such as vascular endothelial growth factor (VEGF) or endothelial cell death by releasing reactive oxygen species and factors including matrix metalloproteinases (MMPs)214,215. In the bloodstream, cancer cell distribution is determined by blood flow and interactions between cancer cells and the secondary organs that they colonize: cells can get trapped in narrow capillary beds, such as those of the lung and liver, and can also express receptors that bind to metastasis-supporting sites (d) or to platelets (e), which protect the cancer cells from the immune system216–220. Cancer cells can circulate for more than 2 hours, suggesting that they do not always become lodged in the first capillary beds that they reach221. After reaching the secondary site, cancer cells can exit the bloodstream (f) by inducing endothelial cell retraction or death222,223. To proliferate in the secondary site, cancer cells co-opt the local environment by releasing pro-inflammatory compounds and proteinases that induce their neighbours to release growth factors224,225 (g).

REVIEWS

NATURE REVIEWS | CANCER VOLUME 12 | JANUARY 2012 | 41

© 2012 Macmillan Publishers Limited. All rights reserved

Nature Reviews Cancer 2012, 12, 39–50

Emerging Frontiers in Drug DeliveryMark W. Tibbitt,†,‡ James E. Dahlman,§,∥ and Robert Langer*,†,‡,⊥

†Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts 02142, UnitedStates‡Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02142, United States§Broad Institute of MIT and Harvard, Cambridge, Massachusetts 02142, United States∥Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology, Atlanta, Georgia 30332, United States⊥Harvard-MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, Massachusetts02139, United States

ABSTRACT: Medicine relies on the use of pharmaco-logically active agents (drugs) to manage and treat disease.However, drugs are not inherently effective; the benefit ofa drug is directly related to the manner by which it isadministered or delivered. Drug delivery can affect drugpharmacokinetics, absorption, distribution, metabolism,duration of therapeutic effect, excretion, and toxicity. Asnew therapeutics (e.g., biologics) are being developed,there is an accompanying need for improved chemistriesand materials to deliver them to the target site in the body,at a therapeutic concentration, and for the required periodof time. In this Perspective, we provide an historicaloverview of drug delivery and controlled release followedby highlights of four emerging areas in the field of drugdelivery: systemic RNA delivery, drug delivery for localizedtherapy, oral drug delivery systems, and biologic drugdelivery systems. In each case, we present the barriers toeffective drug delivery as well as chemical and materialsadvances that are enabling the field to overcome thesehurdles for clinical impact.

1. INTRODUCTIONMedicine relies on the use of pharmacologically active agents(therapeutics or drugs) to manage or reverse the course ofdisease. The current global pharmaceutical market is valued at$980 billion annually, and, in the U.S., nearly 50% of thepopulation has used at least one prescription medication in thepast 30 days.1,2 Notably, pharmacologically active agents arenot inherently effective; their benefit is directly coupled to themanner by which they are administered. Administration affectsdrug pharmacokinetics (PK), absorption, distribution, metab-olism, duration of therapeutic effect, excretion, and toxicity.3 Asnew therapeutic molecules are discovered, there is anaccompanying need for improved modes of delivery, and aclearer scientific understanding of how drug administrationaffects safety and efficacy.In the ideal case, drugs would be applied in vivo at exactly the

therapeutic concentration and would precisely target cells thatcause disease. However, drug delivery is not easily controlled.Drug release rates, cell- and tissue-specific targeting, and drugstability are difficult to predict. To address these limitations,drug delivery systems (DDS) have been designed using a wide

array of materials and chemical strategies. Here, we define DDSas technologies that are designed to improve the specificity oftherapeutics by stabilizing them in vivo, controlling their release,and localizing their effect. Many materials have releasedtherapeutics for prolonged periods of time and at targetedlocations within the body; the properties of DDS are tailored tothe physicochemical attributes of the drug and the intendedroute of administration (Figure 1). DDS have been propelled

by advances in synthetic chemistry, materials science, medicalchemistry, and conjugate chemistry, and are growingincreasingly common in the clinic. However, the field ofmedicine is in active transformation as therapies based onnucleic acids, antibodies, proteins, and drug conjugates emerge.The translation of these therapeutic molecules, which can beorders of magnitude larger than therapeutic small molecules,and significantly more sensitive to environmental effects, willrequire adequate protection, bioavailability, and specificity. As aresult, DDS will need to evolve. In this Perspective, we first

Received: September 22, 2015

Figure 1. Hurdles to delivery and DDS design criteria vary with routeof administration. Drugs can be administered in a variety of ways, andtheir successful delivery requires different design criteria. For example,systemic delivery requires the drug to avoid clearance by thereticuloendothelial system, and enter the correct tissue. DDS forlocal delivery must avoid damage to the surrounding tissue, and mustcontrol release to prevent dose “dumping”. Oral delivery systems mustovercome extreme changes in pH as well as accommodate changes inbiomolecule concentrations that vary with food intake.

Perspective

pubs.acs.org/JACS

© XXXX American Chemical Society A DOI: 10.1021/jacs.5b09974J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Various Routes of Drug Administration:

Robert Langer, et al. JACS 2016,138,704−717

Traditional ways to treating cancer;

ChemotherapyUse of anti-cancer (cytotoxic) drugs to destroy cancer cells. Work by disrupting the growth of cancer cells !Non specificity !Toxicity !Adverse side effects !Poor solubility

Background and Introduction¾ Cancer

Development of abnormal cells that divide uncontrollably which have the ability to infiltrate and destroy normal body tissue

¾ Chemotherapy

�Nonspecificity � Toxicity� Adverse side effects� Poor solubility

Use of anti-cancer (cytotoxic) drugs to destroy cancer cells.

Work by disrupting the growth of cancer cells

How Is Radiation Therapy Used?How Is Radiation Therapy Used?

Radiation therapy is used Radiation therapy is used

two different ways.two different ways. To cure cancer:To cure cancer:

• Destroy tumors that have not Destroy tumors that have not

spread to other body parts.spread to other body parts.

• Reduce the risk that cancer will Reduce the risk that cancer will

return after surgery or return after surgery or

chemotherapy.chemotherapy. To reduce symptoms:To reduce symptoms:

• Shrink tumors affecting quality of Shrink tumors affecting quality of

life, like a lung tumor that is life, like a lung tumor that is

causing shortness of breath.causing shortness of breath.

• Alleviate pain by reducing the Alleviate pain by reducing the

size of a tumor.size of a tumor.

Effective drug delivery system

Retain

Evade Target

Release

Before developing an understanding of how increas-ing the macromolecular complexity can improve the delivery of active therapeutic agents, it may be advan-tageous to briefly review the basic requirements of designing a nanocarrier for systemic delivery19–21. First, the nanocarriers should be able to carry a sufficient dose of a hydrophobic or hydrophilic drug and remain in cir-culation in a physiological medium with tunable leakage. Second, it should target, accumulate and distribute at the desired site in anatomic as well as subcellular com-partments. Last, it should be biocompatible. In simplistic terms, a drug delivery vehicle has to solubilize the active pharmaceutical agent, tailor interactions with cells to maximize drug uptake by mechanisms that include, for example, adsorption or endocytosis, and minimize elim-ination or degradation of its contents before reaching its target. In addition, the drug delivery vehicle should be non-immunogenic, with a cost-effective synthesis that is easy to scale up.

Drug delivery systemsThere has been significant effort devoted to designing effi-cient nanodevices (for example, liposomes, niosomes and solid-lipid nanoparticles) for delivering pharmaceutical agents. Although not strictly macromolecular carriers,

liposomes are some of the most extensively investigated drug delivery vehicles and have seen much success in clinics. It is thus appropriate to briefly discuss their use before the discussion of macromolecular nanocarriers.

LiposomesLiposomes, formed by the self-assembly of amphiphilic phospholipids, have been explored for drug delivery for more than 50 years22. These thermodynamically stable spheres can encage both hydrophilic and hydro phobic drugs, and have become established for their use in clinically approved formulations23. Liposomes have offered distinct advantages and considerable prom-ise in delivering a plethora of otherwise inefficient drugs by modifying their physicochemical properties and biodistribution, and by reducing drug toxicities24. PEGylation (PEG, poly(ethylene glycol)) is a commonly adopted technique for imparting stealth properties, and PEGylated liposomal nanoparticles have generally been considered to be benign and inert carriers25. However, it has been found that PEG itself can elicit an immuno-genic response26. More recently, anti-PEG antibodies have been described as a new platform that can further enhance the efficacy of liposomal formulations27,28 by targeting the liposomes towards specific cell types.

Accelerated clinicalvalidation by matchmaking

1960

1970

1971

1980

1981

1990

1991

2000

2001

2010

2011

2017

Nanocarrier therapeutics• Easy development, ability to tune

binding of nanocarriers to biological substrates

• Area is still in its infancy, with more work required to decode its full potential

Linear polymers• Synthetic articulation, targeting,

environment and stimuli responsive, improved pharmacokinetics and pharmacodynamics

• Heterogeneity, unstable self-assemblies, conjugation irreproducibility, high immunogenicity

Miktoarm polymers• Compact architecture, lower CMCs,

increased drug encapsulation in stable aggregated assemblies, multifuctional

• Complex synthesis, limited data and characterization

Liposomes• Enhanced aqueous circulation half-lives, PEGylation reduces clearance• Poor drug loading, leakage, targeting, immunogenicity, biological fate of phospholipids

Dendrimers• Homogeneity, reproducibility, tailored multivalency, higher permeation and circulation• Limited synthetic versatility, spatial distribution of functions, physicochemical characterization

Telodendrimers• Combined advantages of two architectures, tailored incorporation of multitasking units• Early stages of development

Figure 1 | Analysis of macromolecular structure evolution in drug delivery. Nanomaterials have had a key role in delivering active pharmaceutical agents to the diseased site. Here, we provide a brief summary of the timeline, advantages and disadvantages of each category of nanocarriers, ranging from early discoveries of phospholipids and linear polymers to branched and hyperbranched macromolecules, hybrids thereof, drug-free macromolecules as therapeutics themselves, and strategies to accelerate bench-to-bedside transition. CMC, critical micelle concentration; PEG, poly(ethylene glycol).

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2 | ARTICLE NUMBER 0063 | VOLUME 1 www.nature.com/natrevchem

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History of Targeted Drug delivery systems:

Nature Reviews, 2017, 1 (63), 1-17.

TYPES OF DRUG DELIVERY

TUMOR-TISSUE TARGETINGEnhanced permeability and retention (EPR) 1.Consequence of tumor angiogenesis. 2. Due to rapid proliferation of endothelial cells during angiogenesis usually results in a reduced density of endothelial cells. 3.Thus loss of tight junctions and formation of large gaps between the cells 4.Furthermore, tumor tissues usually lack effective lymphatic drainage 5.The EPR effect is important for nanoparticle delivery to cancer tissue

OpsoninsPlasma proteins (for example, immunoglobulins, complement proteins and fibrinogen) that coat a foreign particle to facilitate its uptake and destruction by phagocytic cells.

Mononuclear phagocyte system(MPS). Part of the immune system composed of scavenging monocytes and macrophages, located in reticular connective tissue surrounding, for example, the liver, spleen, lung and bone marrow.

growth factor (HGF)), proteins and peptides (for exam-ple, endostatin and tumstatin), and endothelial cells and endothelial progenitor cells, have been described75,76 . However, their role, along with other potential bio-markers in predicting EPR, needs further investigation in preclinical and correlative clinical studies.

Enhancing drug delivery to the tumourNP–protein interactions. When a NP enters a biologi-cal environment (for example, blood, interstitial fluid or extracellular matrix (ECM)), its surface is rapidly cov-ered by various biomolecules (typically proteins), leading to the formation of a ‘corona’ (REFS 77–81) (FIG. 2b). The adsorption of proteins alters the particle size, stability and surface properties and, more importantly, provides the NPs with a biological identity that determines the physio-logical responses they elicit, ranging from cellular uptake and intracellular trafficking to PK, biodistribution and

toxicity (FIG. 2c–f). For instance, the binding of opsonins can trigger recognition and clearance by the mononuclear phagocyte system (MPS)79. Conversely, it has also been suggested that a corona rich in dysopsonin proteins (for example, apolipoproteins and albumin), which inhibit phagocytic uptake, could contribute to the stealth effect of NPs82–84. While ligand-functionalized NPs might lose targeting capability when a protein corona forms on their surface85, decoration of NPs with some particular plasma proteins could improve delivery to specific organs. One recent example is the finding that apolipoprotein E is essential for some siRNA lipoplexes to target hepatocytes in vivo86 . In contrast, NP–protein interactions in clini-cal settings can also trigger hypersensitivity reactions in patients by activating the complement system87.

Using various analytical techniques, several studies have extensively characterized the protein corona (for example, its composition, density, conformation, thick-ness, affinity and dynamics) on certain nanomaterials (for example, gold, silica and polystyrene NPs and lipo-somes)88. It is now clear that NP–protein interactions are highly dependent on the NP physicochemical properties, exposure time as well as protein source and concentra-tion. However, we still do not have a clear picture of how NP properties (FIG. 2a) and protein adsorption patterns (FIG. 2b) correlate with specific physiological responses (FIG. 2c–f). With high-throughput characterization of the serum protein corona fingerprint in a library of 105 different gold NPs, a quantitative multivariate model was developed to predict interaction of NPs with cells89. Protein corona fingerprints and physicochemical prop-erties of 17 liposomal formulations were recently used to predict multiple biological interactions including cellular uptake and viability of various tumour cells90. Attention was also paid to the crucial role of human disease type on the composition of the protein corona and its effects on cellular uptake and toxicity of NPs91. Nevertheless, most of these studies were focused on NP–protein inter-actions in vitro, and little effort has been made to study protein corona formation in vivo and its correlation with PK, biodistribution and therapeutic efficacy. It is note-worthy that the very few in vivo evaluations of the pro-tein corona demonstrated significant differences between in vitro and in vivo results92.

Moreover, we think that this field could be further advanced by addressing the following questions. Do we need specific protein-knockout mouse models to vali-date and explain the observations from in vitro studies and normal mice? In addition to the widely studied pro-teins in serum, how do the proteins in other biological environments, such as the TME, affect the corona, NP interactions with tumour cells and NP penetration across the tumour ECM? What new techniques will we need to more precisely characterize and quantify the in situ pro-tein corona? We expect that by extending the methodo-logy of quantitative structure–activity relationships to diverse NP platforms and biological responses, such nanomics approaches could facilitate a deeper under-standing and better control of the nano–bio interface and prompt more rational design of safe, effective and even patient-specific nanomedicines.

Nature Reviews | Cancer

Patients with heterogeneous tumour EPR effect

Potential EPR markers

Patients with high EPR effect to receive nanotherapeutics

Companion imaging NPs Theranostic NPs Serum or tissue biomarkers

Pros• No modi cation of

therapeutic NPs• Proof‑of‑concept

available in animal models and in patients

• Non‑invasive imaging

Cons• Regulatory, marketing

and use complexity

Pros• More precise tracking• Proof‑of‑concept

available in animal models and in patients

• Non‑invasive imaging

Cons• Complexity of chemistry

and manufacturing

Pros• Detection using

patient samples• Proof‑of‑concept

available in animalmodels

Cons• Require serum sample

or tumour biopsy• Need more biological

understanding

Fe3O

4

ba c

Figure 3 | Potential markers for predicting EPR effect and nanotherapeutic efficacy. a | Companion imaging agents (for example, ferumoxytol nanoparticle (NP)) have been applied to predict the accumulation of poly(d,l‑lactic-co-glycolic acid)-b-poly(ethylene

glycol) (PLGA-PEG) NP-encapsulated docetaxel and its anticancer activity in solid

tumours, and ferumoxytol is currently in clinical trials to determine its feasibility as a

predictive marker for the liposomal irinotecan MM-398. b | Therapeutic NPs labelled with imaging agents (for example, radioisotopes), also called theranostic NPs, have been used

to monitor their biodistribution and tumour accumulation using various imaging

techniques both preclinically and clinically. c | Serum and tissue biomarkers may also serve as surrogate markers for the enhanced permeability and retention (EPR) effect, as suggested by one recent example showing strong correlation of liposome accumulation

in tumours with the relative ratio of matrix metalloproteinase 9 (MMP9) to tissue inhibitor

of metalloproteinase 1 (TIMP1) in the circulation.

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Nature Reviews Cancer, 2017, 17, 20–37

DIFFERENT PHARMACEUTICAL CARRIERS

form offers many attractive features, including: 1) improveddelivery of drugs that are poorly soluble in water and deliveryof a therapeutic agent into cancerous cells at a high dose;2) better protection of a drug from harsh environments (e.g.,

the highly acidic environment in the stomach or the lysosomesof a cell, and the high levels of proteases or other enzymes inthe blood stream) before they can reach the targets, leading toan extended plasma half-life of the drug in the systemic

Younan Xia studied at the University ofScience and Technology of China (B.S. in1987) and UPenn (M.S. in 1993), andreceived his Ph.D. from Harvard in 1996(with George Whitesides). He started asAssistant Professor at the University of Wash-ington (Seattle) in 1997, and was promotedto Associated Professor and Professor in2002 and 2004, respectively. He joined theDepartment of Biomedical Engineering atWashington University in St. Louis in 2007as the James M. McKelvey Professor. In early2012, he moved to Georgia Tech to take the

position of Brock Family Chair and GRA Eminent Scholar in Nano-medicine. His research interests include nanomaterials, biomaterials, nano-medicine, and regenerative medicine.

Tianmeng Sun has been a postdoctoralfellow in the Xia group at Georgia Techsince August 2012. He received both hisB.S. (in 2006) and Ph.D. (in 2011) in lifescience and cell biology from the Universityof Science and Technology of China. Histhesis focused on the development of nano-particular delivery systems for cancer therapybased on siRNAs and chemotherapeuticagents. His current research interests focuson the development of nanomaterials forcancer and atherosclerosis treatments.

Yu Shrike Zhang received his B.S. in Bio-medical Engineering from Southeast Univer-sity, China, in 2008. He received his Ph.D.in biomedical engineering (with Prof.Younan Xia) in 2013, and then joined Prof.Ali Khademhosseini’s group as a postdoctoralfellow at Harvard Medical School, Brighamand Women’s Hospital, and Harvard-MITDivision of Health Sciences and Technology.His research interests include biomaterials,tissue engineering, regenerative medicine,imaging, nanomedicine, and lab-on-a-chip.

Bo Pang received his B.E. in mechanicalengineering (2010) and B.S. in chemistry(2011) from Peking University, China. Cur-rently, he is pursuing his Ph.D. degree inbiomedical engineering in the joint Ph.D.program of Georgia Tech/Emory/PekingUniversity under the supervision of Profs.Qiushi Ren and Younan Xia. His researchinterests focus on the use of gold nanocagesfor cancer therapy and nanoparticle-basedimaging contrast agent for PET and SPECT.

Figure 1. A summary of nanoparticles that have been explored as carriers for drug delivery in cancer therapy, together with illustrations ofbiophysicochemical properties.

.AngewandteReviews Y. Xia et al.

&&&& www.angewandte.org ! 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Angew. Chem. Int. Ed. 2014, 53, 2 – 47! !

These are not the final page numbers!

Nanoparticles explored as carriers for drug delivery in cancer:

Angew. Chem. Int. Ed. 2014, 53, 2–47

Primary Trageting:

Phage displayA selection technique in which a library of peptide or protein variants is expressed on the outer membrane of virus-infected bacteria (phage virion) and then screened for binding affinity using a process called panning.

Ribosome displayA selection technique in which diverse gene sequences encoding functional proteins are produced by ribosomes and then screened for their affinity to bioactive targets using a process called panning.

AptamersOligonucleotides with high binding affinity to proteins or other molecules.

Peptide nucleic acidsArtificial polymers that mimic the DNA or RNA base structure, but that replace the negatively charged deoxyribose and ribose sugar backbone with N-(2-aminoethyl)-glycine units linked by peptide bonds.

Once at the target organ, steering nanoparticles towards the malignant cells poses an additional challenge. This has been addressed using several approaches. One approach is to use external driving forces, such as mag-netic fields to concentrate iron oxide nanoparticles114,115, or acoustic waves to trigger micro-bubble localization116. Recently, an active nano-signalling system was devel-oped, in which one targeted nanomaterial triggers a local biological cascade that, in turn, recruits other therapeutic nanoparticles to the disease site117. This ability to amplify a local signal may be especially important for locating and treating metastases. Another mechanism for steering nanomaterials to a disease site is by using self-propelled nanoparticles that can navigate autonomously118,119.

Secondary targeting — the cancer cell. Targeting a metastatic cancer cell, either in transit from the primary tumour or buried within a population of non-cancerous cells, presents a unique challenge. In contrast to primary targeting, secondary targeting is the precise homing of a particle or a drug to a specific cell type (FIGS 2,3). It can require a chemical specificity that enables the nanopar-ticle to bind to unique moieties that are presented by the cancer cell. Cancer cells, whether metastatic or part of the primary tumour, can upregulate certain cell-surface mol-ecules and secreted factors, and may even express proteins that are usually only expressed during embryonic devel-opment120. A metastatic cell will also express endogenous surface proteins from its site of origin, which will differ from its site of implantation. For example, a metastatic pancreatic cancer cell is distinct from cells within the liver strictly by virtue of its pancreatic origin. These character-istics provide investigators with handles to target cancer cells. They may also limit the potential side effects of tar-geting proteins that are expressed by a given cell type that are not exclusive to the cancer cell.

The strategies needed for targeting specific cells (sec-ondary targeting) may differ from those used for target-ing the organ (primary targeting). The researcher must take into account the binding affinity of the nanoparticle to the molecules of interest, as well as binding specific-ity and immunological effects. Antibody conjugates — drug, polymer or radioisotope-labelled antibodies — are currently in the clinic for targeting cancer. For

instance,131I-tositumomab (Bexxar; GlaxoSmithKline) is a combination therapy that involves a radiolabelled CD20-specific antibody for targeting follicular B cell lymphoma121. Antibody-based targeting ligands have been used on various nanodelivery systems122–126. Likewise, short peptides, including those with integrin-binding domains RGD and IKVAV, can be appended to nanoparticles and can increase their binding to specific cell types within a tissue107,127–129.

High-throughput methods, such as phage display, ribosome display, in vitro evolution and in vitro selection are being used to discover new targeting ligands130, such as antibodies, peptides and nucleic acid-based ligands (aptamers)131,132. Pegaptanib, an anti-angiogenic aptamer-based agent, is being used clinically for the treatment of macular degeneration133; however, aptamers have not yet been approved for cancer treatment. Peptide nucleic acids (PNAs) can bind with a high affinity to complimentary DNA strands, and the peptide backbone allows covalent modification with targeting ligands and fluorophores134. PNAs have been used to target pro-metastatic genes and to inhibit their expression135.

Small-molecule-binding domains, such as the folate receptor which is overexpressed in human oral carci-noma, metastatic breast, colorectal and other cancers, are also under investigation and demonstrate affinity to nanoparticles coated with folic acid136–139. Certain cells, such as macrophages, which routinely phagocytose par-ticles, can be targeted with materials, such as dextran78, which resemble lipopolysaccharides that are expressed on the surface of bacteria. Phagosomes tend to fuse with lysosomes, however, resulting in the degradation of the contents; thus, strategies must be used to control the route of uptake140.

The route through which nanoparticles enter a cell can also be engineered, potentially affecting the cellular compartment into which a drug is released. This is an emerging area and will probably evolve into a separate field of tertiary targeting141, because the intracellular fate of the particle can determine the resulting efficacy of the encapsulated drug142. Clathrin-dependent endo-cytosis, one of the most well-characterized pathways of cell uptake, primarily results in entrance to the lysosomal pathway143. This type of endocytosis can be triggered by

Table 1 | Primary targeting — general considerations for nanoparticle delivery to specific organs

Target organ Particle size Surface property Comments

Brain 5–100 nm: uptake efficiency decreases exponentially with size

Lipophilic moieties and neutral charge enhance brain uptake

Leukocytes can take up nanoparticles in circulation and then carry them to disease sites in the brain

Lung >200 nm: particles are trapped in lung capillaries

Positive surface charge Inhaled particles with low density (<0.4 g per cm3) and of large size (>5 mm) are also retained in the lung

Liver <100 nm, to cross liver fenestrae and target hepatocytes. >100 nm particles will be taken up by Kupffer cells

No specificity needed Lipid and lipid-like materials tend to accumulate in the liver

Lymph nodes 6–34 nm: intra-tracheal administration. 80 nm: subcutaneous administration

Non-cationic, non-pegylated and sugar-based particles

200 nm particles in circulation can be taken up by leukocytes and trafficked to lymph nodes

Bone Unknown Compounds such as alendronate and aspartic acid adhere to bone and have been used for bone targeting

Despite great importance, bone targeting is under-researched

 

REVIEWS

NATURE REVIEWS | CANCER VOLUME 12 | JANUARY 2012 | 43

© 2012 Macmillan Publishers Limited. All rights reserved

Nature Reviews Cancer 2012, 12, 39–50

Nanoscale vehicles have been derived from biological, organic and inorganic origins to address a wide variety of biological mechanisms and targets

Tunable imaging agentsThe emission wavelength of quantum dots can be modulated by changing their size.

accumulate in the tumour vasculature during angiogen-esis166 and can deliver a therapeutic payload. pH-sensitive and temperature-sensitive formulations have been devel-oped to control the release of the payload167,168. Synthetic, lipid-like materials that form lipoplexes have been pro-duced by combinatorial techniques for applications such as siRNA delivery88,148.

Gold nanoparticles have been used for thermo ablative therapies41. Gold shells, spheres and rods respond to near-infrared light by releasing energy in the form of heat that induces the coagulation of the tumour vasculature and that can cooperatively increase the therapeutic effect of other targeted therapies169. Gold nanoparticles can also be used as scaffolds to which multiple ligands are attached170. Other nanomaterial classes, such as iron nanoparticles and carbon nanotubes or spheres (buckyballs), have been used to deliver therapies, often by binding the drug to the outer surface or by filling the interior, where applicable171.

Biological response to nanomaterialsDifferent types of materials exhibit varying bio-dis-tribution, compatibility, degradation and circulation properties. No single parameter can be denoted as the most important prerequisite for effective cancer therapy. Recent studies have identified cytokines that are upregu-lated after the administration of positively charged nano-particles104,172. Compliment activation has been associated with nanoparticle administration. Particles with positive surface charge activate the classical compliment pathway, and negatively charged particles activate the alternative (lectin) pathway173,174. Interestingly, it has been shown that different degrees of PEG on the surface of nanopar-ticles affect the complement activation pathways; lower levels of PEG are associated with the classical pathway, while higher degrees of PEG are associated with moderate activation of the lectin pathway175. Particle size also has a role in this process; the larger the nanoparticle, the higher the extent of opsonization176. In many cases, adverse bio-logical responses to nanoparticle administration, such as inflammation or compliment activation, can be treated with pre-therapy or post-therapy medication177.

In an attempt to improve the biocompatibility of nanoparticles in vivo, a hybrid biomimetic approach has been undertaken. Nanoparticles were disguised by coating them with a naturally derived erythrocyte mem-brane (also known as ‘red-blood-cell ghosts’)178–182 or by physically loading the particles into stem cells, thereby evading reticuloendothelial system (RES) clearance and using natural pathways to target cancer180,183,184. A differ-ent approach uses cell membranes as scaffolds for con-structing nanoparticles, using targeting moieties that are naturally present on the cell surface and of the biocom-patibility of biologically derived materials185,186. Taking advantage of the body’s natural trafficking modalities (that is, cells and complex proteins) is a new and prom-ising approach for delivering nanoparticles to specific tissue compartments.

The toxicity of nanomaterials is under investigation; a meta-analysis illustrates that their effect on tissues depends on the physicochemical properties of the materi-als used, including size, charge and coating ligands187. For example, the semiconductor cores of quantum dots can be cytotoxic, but certain polymer coatings have reduced toxic effects in vivo187. Nanoscale gold particles exhibit minimal toxicity on mammalian tissues, but they do not naturally degrade in vivo and can accumulate in organs unless their surface is decorated with stealth materials such as PEG188. Careful engineering of drug carriers can poten-tially reduce the amount of foreign material, both drug and nanoparticle, that is administered to the patient8,148.

Diagnosis and detectionThe treatment of metastatic disease increasingly depends on imaging and diagnostics (FIG. 2; TABLE 2). Some tools, such as directed radiotherapy, require precise tumour localization, and treatment decisions are based on under-standing the extent of disease spread. Diagnostics, such as contrast agents for radioimaging, visualization aids for surgeons and molecularly activated sensors, comprise an active area of investigation for materials engineers work-ing at the nanoscale. Much of the excitement in this area stems from the unique material properties that appear at this scale. For example, the fluorescent properties of highly photostable tunable imaging agents, such as quan-tum dots, only appear when semiconductor crystals are synthesized with nanometer dimensions. For patients with metastatic cancer, the work in this field has the potential to reduce toxicity while increasing the speci-ficity and signal strength of imaging agents; enable the visualization of metastases during surgery; and provide molecular sensors to aid in many areas, from the dosing of chemotherapy to defining the onset of malignancy.

For magnetic resonance imaging (MRI), superpara-magnetic nanoparticles consisting of iron oxide (SPIONs) can yield higher contrast at lower concentrations than gadolinium, a common MRI contrast agent. Such par-ticles that are decorated with dextran, which localize within lymph nodes, have been studied for nodal tumour detection in patients with prostate cancer80. Targeted SPIONs, coated with RGD peptide, have been inves-tigated in vivo to image integrin αVβ3-positive tumour neovasculature189. Nanoparticles have been explored for

Table 2 | Nanoparticulate building blocks and their uses

Building block

Vehicle Uses

Polymers Core-shell nanoparticles, nanogels and polymer micelles

Well-characterized, biocompatible and modular delivery vehicles

Lipids Liposomes, lipoplexes, micelles and filomicelles

Delivers water-soluble and -insoluble drugs effectively

Metals Gold nanorods, gold nanoparticles, iron oxide nanoparticles and quantum dots

Imaging agents for diagnosis. Thermoablative therapies

Carbon Carbon nanotubes, nanodiamonds and graphene

Near-infrared emissions allow for tissue-transparent imaging for diagnosis and tracking. Therapies to potentially sidestep MDR in some leukaemias

Biologicals Viruses, nucleic acid nanoparticles, DNA origami and protein nanoparticles

Viruses deliver a non-covalently bound payload without loss from passive diffusion

MDR, multi-drug resistance.

REVIEWS

46 | JANUARY 2012 | VOLUME 12 www.nature.com/reviews/cancer

© 2012 Macmillan Publishers Limited. All rights reserved

Nature Reviews Cancer 2012, 12, 39–50

1.Doxorubicin interacts with DNA by intercalation and inhibition of macromolecular biosynthesis. 2.This inhibits the progression of topoisomerase II, an enzyme which relaxes supercoils in DNA for transcription.

Doxorubicin Mechanism of action:

https://en.wikipedia.org/wiki/Doxorubicin

Structure of Doxil

Doxorubicin

Lipid Membrane (Phospholipid +

Cholesterol)

Polyethylene Glycol

85-100 nm

19

Slide 17

Doxorubicin Levels in Prostate Carcinoma Xenograft

Vaage J, et al. Cancer, 1994

µg D

rug/

gm T

umor

Hours100 150 20050

Doxil

Adriamycin

4

6

8

2

0

Doxil AUC = 919

Adriamycin AUC = 36.5

Slide 16

Plasma Levels of Total and Liposome-Encapsulated Doxorubicin after Doxil

Days After Infusion0 1 2 3 4 5 6 7

0.25

0.50

2.50

5.00

25.00

1.00

10.00 Total Doxorubicin

Encapsulated Doxorubicin

Dox

orub

icin

(Pg

/mL)

Single 50 mg/m2 dose of DoxilMean values ± SD for 14 patients, mixed histologiesGabizon et al, Cancer Res (1994)

The therapeutic benefits of these new drug delivery systems include:

1. Increased efficacy of the drug. 2. Site specific delivery 3. Decreased toxicity/side effects 4. Increased convenience 5. Viable treatments for previously incurable

diseases 6. Potential for prophylactic applications 7. Better patient compliance.

•Warburg hypothesis: Cancer cells undergo higher than usual rates of intracellular glycolysis despite presence of oxygen – “aerobic glycolysis”

•Approximately 10 times higher than normal cell.

•Increased glycolysis is followed by increased cellular fermentation decreasing oxidative phosphorylation (2ATP vs 38ATP)

•Warburg’s research revolutionised the way cancer cells are differentiated from normal cells

•Increased fermentation: positive feedback ●Lactic acid builds up intracellular pH decrease ●Continues to increase glycolysis + cancer development

- Term comes from the Otto Warburg

- German physiologist and medical doctor

- Awarded Nobel prize in 1931

Science 2009, 324 (5930), 1029-1033.

Warburg Effect:

https://en.wikipedia.org/wiki/Warburg_effect

Positron emission tomography(PET) :A positron emission tomography is a nuclear medical imaging technique which produces a three dimensional image of functional processes in the body.

Br J Clin Pharmacol, 2011, 73 (2), 175–186

1.Based on the detection of radioactivity emitted after a small amount of a radioactive tracer is injected into a peripheral vein 2. The tracer is administered as an intravenous injection usually labelled with oxygen-15, fluorine-18, carbon-11, or nitrogen-13 3. One clinical use of this is to distinguish between benign and malignant tumours

This is the Whole-body PET acquisition of a 79 kg weighting female. Accumulation of the tracer in the heart, bladder, kidneys and brain, liver metastases of a colorectal tumor are clearly visible within the abdominal region of the image.

https://en.wikipedia.org/wiki/Positron_emission_tomography

[18F] 2-fluorodeoxyglucose (FDG):one of the most frequently used PET reagent

Synthesis:

ACS Cent. Sci. 2015, 1, 142−147.

and ready access to a wide variety of nucleophiles in thetransacylation reaction.23−25

As a first step, we generated a short synthetic peptide, (Gly)3-R, where R contains a trans-cyclooctene (TCO) functionalitythat enables a TCO-tetrazine ligation reaction with a 18F-tetrazine. The TCO-tetrazine reaction is fast, with an estimatedsecond order rate constant of 210−26000 M−1 s−1.26−28 Weestablished a method for 18F labeling using commerciallyavailable 18F-FDG, the principal source of 18F in clinical use.The dynamic equilibrium between an aldohexose in its linearaldehyde form (the reactive molecular species) and its cyclicalhemiacetal derivative permits the installation of 18F-FDG on anaminooxy-functionalized molecule.29

In view of the t1/2 of18F ≈ 110 min, any synthetic process

using 18F as a substrate and the necessary downstreampurification steps must be rapid. Thus, we first optimizedreaction conditions using nonradioactive FDG and charac-terized the reaction products by liquid chromatography−massspectrometry (LC−MS) (Supporting Information). Severaldifferent catalysts have been reported for the oxime ligationreaction, of which the phenylenediamines are among the mostefficient. While m-phenylenediamine is a more efficient catalystthan p-phenylenediamine (pPDA), its Schiff base is more stableand can block oxime formation if its concentration relative tothe aminooxy or aldehyde is high.30,31 In our case, theconcentration of aldehyde (18F-FDG) is extremely low (<nM).We therefore used pPDA as the catalyst at ∼0.4−0.6 M andtetrazine-aminooxy in the ∼0.2−0.3 M range. We incubated the

aminooxy-tetrazine with fluorodeoxyglucose in the presence ofthe catalyst, pPDA, with constant agitation at 75 °C for ∼5−10min. High-performance liquid chromatography (HPLC) of thereaction mixture showed (near)-complete consumption ofFDG (Supporting Information). To produce the radioactiveaminooxy-tetrazine derivative, we performed the incubationwith 18F-FDG in the presence of the catalyst, pPDA, withconstant agitation at 75 °C for ∼5−10 min. Radio-HPLCshowed that the coupling reaction with 18F-FDG proceededrapidly, yielding >90% oxime 18F-FDG-tetrazine in ∼5−10 min(Figure 1). We separated the 18F-oxime product by HPLC,followed by capture of the product via a Sep-pak C18 column.A solution containing the TCO-labeled protein of interest,prepared previously using sortase, was then added to thepurified oxime 18F-FDG-tetrazine. The reaction was allowed toproceed for ∼15−20 min at 25 °C with constant agitation. The18F-labeled protein was purified by size exclusion in phosphatebuffer, providing the final 18F-labeled protein ready forinjection.We previously used a 18F-TCO-tetrazine to label proteins

with 18F to image lymphoid organs using an anti-Class II MHCsingle domain antibody, VHH7.32 We evaluated the presentlabeling method to confirm that the binding site of thenanobody remained intact. 18F-VHH7, produced as describedabove, detected secondary lymphoid organs exactly asreported32 (Figure 1 and movie 01 in the SupportingInformation).

Figure 1. (A−C) Site-specific 18F-labeling of proteins using 18F-FDG and sortase. (A) A tetrazine-aminooxy and 18F-FDG were combined in thepresence of p-phenylenediamine to produce 18F-tetrazine. Dynamic equilibrium between hemiacetal and linear forms of the aldohexose allowscapture of the FDG into a tetrazine molecule via an oxime ligation; the 18F-tetrazine product is purified via HPLC. (B) A single domain antibodyfragment (VHH) equipped at its C-terminus with the LPXTG sortase-recognition motif is site-specifically modified with a (Gly)3-trans-cyclooctene(TCO), as confirmed by LC-MS (Supporting Information). (C) 18F-Tetrazine was added to the TCO-modified VHH, and after ∼20 min the labeledVHH was retrieved by rapid size exclusion chromatography. (D−F) 18F-VHH7 (anti-mouse class II MHC) detects secondary lymphoid organs. (D)PET images of a representative C57BL/6 mouse 2 h postinjection of 18F-VHH7; numbers indicate (i) lymph nodes: 1, 2, 3, 4, 7, 8, 9; (ii) thymus: 5;(iii) spleen: 6. (E) PET-CT images of C57BL/6 mouse imaged with 18F-VHH7 from two different viewpoints (top and bottom panels); clearlylymph nodes and thymus are visible. See movie 01 in Supporting Information for a 3D visualization of lymphoid organs. (F) PET signals in vivo indifferent organs. Experiments are representative of three mice with similar results.

ACS Central Science Research Article

DOI: 10.1021/acscentsci.5b00121ACS Cent. Sci. 2015, 1, 142−147

143

Proposed scheme:

and ready access to a wide variety of nucleophiles in thetransacylation reaction.23−25

As a first step, we generated a short synthetic peptide, (Gly)3-R, where R contains a trans-cyclooctene (TCO) functionalitythat enables a TCO-tetrazine ligation reaction with a 18F-tetrazine. The TCO-tetrazine reaction is fast, with an estimatedsecond order rate constant of 210−26000 M−1 s−1.26−28 Weestablished a method for 18F labeling using commerciallyavailable 18F-FDG, the principal source of 18F in clinical use.The dynamic equilibrium between an aldohexose in its linearaldehyde form (the reactive molecular species) and its cyclicalhemiacetal derivative permits the installation of 18F-FDG on anaminooxy-functionalized molecule.29

In view of the t1/2 of18F ≈ 110 min, any synthetic process

using 18F as a substrate and the necessary downstreampurification steps must be rapid. Thus, we first optimizedreaction conditions using nonradioactive FDG and charac-terized the reaction products by liquid chromatography−massspectrometry (LC−MS) (Supporting Information). Severaldifferent catalysts have been reported for the oxime ligationreaction, of which the phenylenediamines are among the mostefficient. While m-phenylenediamine is a more efficient catalystthan p-phenylenediamine (pPDA), its Schiff base is more stableand can block oxime formation if its concentration relative tothe aminooxy or aldehyde is high.30,31 In our case, theconcentration of aldehyde (18F-FDG) is extremely low (<nM).We therefore used pPDA as the catalyst at ∼0.4−0.6 M andtetrazine-aminooxy in the ∼0.2−0.3 M range. We incubated the

aminooxy-tetrazine with fluorodeoxyglucose in the presence ofthe catalyst, pPDA, with constant agitation at 75 °C for ∼5−10min. High-performance liquid chromatography (HPLC) of thereaction mixture showed (near)-complete consumption ofFDG (Supporting Information). To produce the radioactiveaminooxy-tetrazine derivative, we performed the incubationwith 18F-FDG in the presence of the catalyst, pPDA, withconstant agitation at 75 °C for ∼5−10 min. Radio-HPLCshowed that the coupling reaction with 18F-FDG proceededrapidly, yielding >90% oxime 18F-FDG-tetrazine in ∼5−10 min(Figure 1). We separated the 18F-oxime product by HPLC,followed by capture of the product via a Sep-pak C18 column.A solution containing the TCO-labeled protein of interest,prepared previously using sortase, was then added to thepurified oxime 18F-FDG-tetrazine. The reaction was allowed toproceed for ∼15−20 min at 25 °C with constant agitation. The18F-labeled protein was purified by size exclusion in phosphatebuffer, providing the final 18F-labeled protein ready forinjection.We previously used a 18F-TCO-tetrazine to label proteins

with 18F to image lymphoid organs using an anti-Class II MHCsingle domain antibody, VHH7.32 We evaluated the presentlabeling method to confirm that the binding site of thenanobody remained intact. 18F-VHH7, produced as describedabove, detected secondary lymphoid organs exactly asreported32 (Figure 1 and movie 01 in the SupportingInformation).

Figure 1. (A−C) Site-specific 18F-labeling of proteins using 18F-FDG and sortase. (A) A tetrazine-aminooxy and 18F-FDG were combined in thepresence of p-phenylenediamine to produce 18F-tetrazine. Dynamic equilibrium between hemiacetal and linear forms of the aldohexose allowscapture of the FDG into a tetrazine molecule via an oxime ligation; the 18F-tetrazine product is purified via HPLC. (B) A single domain antibodyfragment (VHH) equipped at its C-terminus with the LPXTG sortase-recognition motif is site-specifically modified with a (Gly)3-trans-cyclooctene(TCO), as confirmed by LC-MS (Supporting Information). (C) 18F-Tetrazine was added to the TCO-modified VHH, and after ∼20 min the labeledVHH was retrieved by rapid size exclusion chromatography. (D−F) 18F-VHH7 (anti-mouse class II MHC) detects secondary lymphoid organs. (D)PET images of a representative C57BL/6 mouse 2 h postinjection of 18F-VHH7; numbers indicate (i) lymph nodes: 1, 2, 3, 4, 7, 8, 9; (ii) thymus: 5;(iii) spleen: 6. (E) PET-CT images of C57BL/6 mouse imaged with 18F-VHH7 from two different viewpoints (top and bottom panels); clearlylymph nodes and thymus are visible. See movie 01 in Supporting Information for a 3D visualization of lymphoid organs. (F) PET signals in vivo indifferent organs. Experiments are representative of three mice with similar results.

ACS Central Science Research Article

DOI: 10.1021/acscentsci.5b00121ACS Cent. Sci. 2015, 1, 142−147

143

(A) A tetrazine-aminooxy + 18F-FDG were combined in the presence of p-phenylenediamine to produce 18F-tetrazine.

(B) A single domain antibody fragment (VHH) equipped at its C-terminus with the LPXTG sortase-recognition motif is site-specifically modified with a (Gly)3-trans-cyclooctene (TCO).

(C) 18F-Tetrazine was added to the TCO-modified VHH.

(D) PET images of a representative C57BL/6 mouse 2 h post-injection of 18F-VHH7; numbers indicate (i) lymph nodes: 1, 2, 3, 4, 7, 8, 9; (ii) thymus: 5; (iii) spleen: 6.

(E) PET-CT images of C57BL/6 mouse imaged with 18F-VHH7 from two different viewpoints.

(F) PET signals in vivo in different organs.

ACS Cent. Sci. 2015, 1, 142−147.

My Proposal: Use of doxorubicin coupled to FDG and use of PET for continuous evaluation

4. Doxorubicin- Mammalian DNA topoisomerase II is a nuclear enzyme that alters the topological state of DNA and is essential for cell replication and viability. Doxorubicin is a non-selective class I anthracycline agent (is toxic even to healthy cells) whose mode of action includes intercalation between adjacent base pairs of the DNA double helix and binding to topoisomerase to prevent DNA replication.[9][8]

MY PROPOSAL

I propose the use of doxorubicin bound to FDG through an oxime linkage as detailed in fig-iv and use PET for its periodic evaluation (PET scans generally take only about 10 minutes).

Fig-(iv) FDG-Doxorubicin complex

(i)  FDG binds to cancerous cells and as a result doxorubicin is delivered more to the cancerous cells (brings in site-specificity).

(ii)  We can simultaneously evaluate the volume of the cancerous cells and follow its progress (by PET) using only a single compound.

(iii)  The half-life of radioactive 18F is only 110 minutes. The by-product of positron emission- 18O is not radioactive and can be adsorbed by the body. Thus, the harmful radioactive element, that may initiate further mutation, degrades fast, causing least toxicity.

Advantages :

Limitations:

1. The drug has to be administered locally and due to its short half-life, it has to be given quite frequently for continuous evaluation. 2. This treatment may be limited to non-diabetic patients.