Stimuli responsive PEGylated nano assemblies for cancer targeted drug delivery

Using targeted nanoparticles to deliver chemotherapeutic agents in cancer therapy offers many advantages to improve drug delivery and to overcome many problems associated with convention

A dissertation for the degree of doctor of philosophy

Stimuli responsive PEGylated nano-assemblies for

cancer-targeted drug delivery

Department of Molecular Science and Technology

The Graduate School of Ajou University

Dai Hai Nguyen

Acknowledgement

I wish to express in this part my gratitude to the scientists, technicians and other people who were directly and indirectly involved in this work, without the help of whom the findings of this thesis surely could not have been done

First and foremost, I would like to extend immeasurable gratitude to Professor Ki Dong Park, for giving me the opportunity to do my PhD thesis under his supervision I greatly appreciated his supervision for teaching, advising and supporting me throughout

my work I am very grateful for his extreme patience and encouragement during the most stressful time when my results were not good He is a respectable mentor who has kindly supported me in the name of family It was an honor to work under his supervisor

I am grateful to my thesis committee members, Professor Sung-Hwa Yoon, Professor Won-Hee Suh at Ajou University, Professor Ji Hoon Jeong at Sungkyunkwan University, Dr In Kwon Jung at Genoss Company for their numerous suggestions and helpful advice This is a good opportunity to express my gratitude to Professors at Ajou University whose teaching and advice helped me to complete my PhD coursework

I would especially like to thank Dr Yoon Ki Joung who has supported for me for about three years He kindly and friendly guided me from laboratory studies to routine life in Korea I also have deep gratitude towards Dr Jin Woo Bae for being a great mentor His scientific comments are always useful in doing experiments, preparing presentation, and writing a scientific paper

I would like to thank my Vietnamese Professors Thi Phuong Thoa Nguyen, Thi Kieu Xuan Huynh, and Huu Khanh Hung Nguyen for giving this opportunity to me, who taught me fundamental knowledge of chemistry at University of Science-HCMC

I especially appreciate all supports of my past and current members in Biomaterial and Tissue Engineering Laboratory: Dr Kyoung Soo Jee, Dr Jin Woo Bae, Dr Dong Hyun Go, Dr Jung Seok Lee, Dr Kyung Min Park, Dr Se Jin Son, Dr Ngoc Quyen Tran,

Dr Eugene Lih, Jong Hoon Choi, Yeo Jin Jun, In Kyu Hwang, Bae Young Kim, Ji Ho Heo, Seung Soo You, Ki Seong Ko, Ji Hye Oh, Seung Mee Hyun, Dong Hwan Oh, Joo Young Son, Yun Ki Lee, Ji Ho Kim, Min Yong Eom, Thi Thai Thanh Hoang, Thi Phuong

Le I hope all members in BT Lab will obtain the outstanding achievement in your dream and get the happiness in their life

I appreciate all help of my Vietnamese best friends in Korea, Minh Dung Truong, Van Thinh Nguyen, Dinh Chuong Pham, Ngoc Hoi Nguyen, Thanh Quy Nguyen, Hung Cuong Dinh, Thi Hiep Nguyen, Chan Khon Huynh, who helped in several experiments such as XRD, AFM, DLS, Confocal, FACS, cell culture, and animal studies Without them this thesis surely would not have been so multifaceted and prolific I also would like

to be thankful to Korean friends in School of Engineering, Medicine School for your help and support me during my stay here Good luck to all of them

Korean life could be some times stressful and tough, with all the competitiveness and perfectionism Luckily, I have had extensive care, support, and help from my family and friends, who shared with me many wonderful and unforgettable moments throughout

my time here I would like to devote this thesis to them with my sincere gratitude

I would like to thank many of my best friends, Hoang Duy Nguyen, Minh Triet Thieu, Hoang Chuong Nguyen, Nhat Nguyen Nguyen, Xuan Huong Ho… With them I shared the first journey to Korea, as well as the sadness of leaving our lovely home and country

All this would not be possible without my loving immediate family For good or for bad, they are the ones who always stand behind me, and let me know that I am not alone Finally, deeply from my heart, I would like to thank my parents who believe and support me at all time

My best regards to all,

Dai Hai Nguyen

Stimuli responsive PEGylated nano-assemblies for

cancer-targeted drug delivery

Supervisor: Professor Ki Dong Park

A Dissertation Submitted to the Graduate Faculty in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy

June 2013 Department of Molecular Science and Technology

The Graduate School of Ajou University

Dai Hai Nguyen

i

Abstract

Cancer is one of the leading causes of death worldwide and chemotherapy is a major therapeutic approach for the treatment which may be used alone or combined with other forms of therapy However, conventional chemotherapy has the potential to harm healthy cells in addition to tumor cells Using targeted nanoparticles to deliver chemotherapeutic agents in cancer therapy offers many advantages to improve drug delivery and to overcome many problems associated with conventional chemotherapy This work covers the general areas of responsive nanocarriers and encompassed methods of fabricating nanocarrier-based drug delivery systems for controlled and targeted therapeutic application

Chapter 1 provides general information of cancer and cancer treatment strategies The recently cancer treatment based on nanocarrier were introduced In addition, the special features as well as requirements of nanoparticles for targeted drug delivery were presented This chapter describes overall objectives of this study with the current status of stimuli-responsive self-assembled nanocarriers for cancer chemotherapy In chapter 2, self-assembled nanogels based on reducible heparin-Pluronic copolymer was developed for intracellular protein delivery Heparin was conjugated with cystamine and the terminal hydroxyl groups of Pluronic were activated with the VS group, followed by coupling of VS groups of Pluronic with cystamine of heparin The chemical structure, heparin content and VS group content of the resulting product were determined by 1H

1

Chapter 1

General introduction

Overall conclusion

Research activity aimed towards achieving specific and targeted delivery of anticancer agents has expanded tremendously in the last 5 years or so with new avenues of directing drugs

to tumors as well as new types of drugs

In this dissertation, we presented how nanoparticles took advantage of these special features and how nanoparticles could act as a vehicle to specifically deliver cancer-fighting drugs

to tumors We have developed three differ drug delivery systems using PEG and its block copolymer for targeted drug delivery The presence of PEG outer shell helps nanoscale carriers

to bypass the RES clearance, thereby prolonging the circulation time in the blood stream Another advantage that could be taken from the stability of PEG-coated nanospheres is the possibility of attaching antibodies or a fragment of them to the surface of the particles, without destabilizing them, in order to achieve site-specific drug delivery, a major challenge for drug administration Ideally, these “magic missiles” would accumulate

at the diseased tissue and locally liberate the necessary amount of drug The drugs can be released at the desired sites of actions by designing environment-sensitive linkers in side structure of nanoparticles where the linkers respond to the extra/intracellular microenvironment

or external stimuli The design of these types of nanoparticles remains a very interesting research area Controlled release of drug at the site of action will enhance the efficacy and reduce the side effect of drug The combination of the use of stimuli-responsive material and targeting moieties will lead to nanoparticles which can be targeted to the side of action and which will deliver the drug

These approach should provide the creative treatment methods have made it to the clinic and hopefully are well on their way to improving the length and quality of life for cancer patients However, it should be noted that extensive preclinical evaluations are required for these types of nanoparticles before they can be considered to use in patients Subjects which have to be evaluated are the pharmacokinetics of drug loaded/conjugated nanoparticles, effect of the surface-located targeting molecules on the opsonization process and blood circulation times as well as the efficacy and toxicity of the nanoparticles in particlular after repeated administration Mechanistic studies of the intracellular drug release from the nanoparticles are also required to further unravel the kinetics of intracellular nanoparticle destabilization and intracellular drug release

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1 Cancer and strategy treatment

Cancer is one of the leading causes of death worldwide (13%) Each year 12.7 million people worldwide are diagnosed with cancer and there are 7.6 million deaths from the disease in 2008 (WHO).1 It is estimated that there are 24.6 million people alive who have received a diagnosis of cancer in the last five years By 2030, the number of new cancer cases is expected to rise to 21.4 million, with 13.15 million cancer deaths.2 Cancer's total economic impact was estimated at $895 billion in 2008, or 1.5% of the world's gross domestic product This cost did not include direct medical costs, which could potentially double the total economic cost, according to Atlanta-based ACS.3

The cancer treatment during the twentieth century was based on surgery, radiation and chemotherapy Of these modalities, surgery is most effective at an early stage of disease progression However, most cancer operations carry a risk of: pain, infection, loss

of organ function Surgery can also cause cancer cells to spread to different sites Radiation while destroying cancer cells also burns, scars, and damages healthy cells, tissues, and organs Initial treatment with chemotherapy and radiation will often reduce tumor size Radiation can cause cancer cells to mutate and become resistant and difficult

to destroy.4 Chemotherapy is drug therapy that can kill these cells or stop them from multiplying However, it involves poisoning the rapidly growing cancer cells and also destroys rapidly growing healthy cells in the bone marrow, gastro-intestinal tract, etc., and can cause organ damage, like liver, kidney, heart and lungs, and so on Moreover, when the body has too much toxic burden from chemo the immune system is either

is a method of delivering medication to a patient in a manner that increases the concentration of the medication in some parts of the body relative to others The goal of a targeted drug delivery system is to prolong, localize, target and have a protected drug interaction with the diseased tissue The conventional drug delivery system is the absorption of the drug across a biological membrane, whereas the targeted release system

is when the drug is released in a dosage form The clinically most relevant drug targeting strategies were summarized in Figure 1.1 The advantages to the targeted release system

is the reduction in the frequency of the dosages taken by the patient, having a more uniform effect of the drug, reduction of drug side effects, and reduced fluctuation in circulating drug levels

4

Figure 1.1 Overview of the clinically most relevant drug targeting strategies (A) Conventional chemotherapy (free drug) (B) passively targeted drug delivery system by virtue of the enhanced permeability and retention (EPR) effect (C) Active drug targeting

to internalization-prone cell surface receptors (over)expressed by cancer cells generally intends to improve the cellular uptake of the nanomedicine systems (D) Active drug targeting to receptors (over)expressed by angiogenic endothelial cells aims to reduce blood supply to tumours (E) Stimuli-sensitive nanomedicines (F) Local drug delivery

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2 Nanocarrier strategies in cancer chemotherapy

The use of nanotechnology in medicine and more specifically drug delivery is set to spread rapidly Currently many substances are under investigation for drug delivery and more specifically for cancer therapy are used in the clinic Interestingly pharmaceutical sciences are using nanocarriers to reduce toxicity and side effects of drugs and up to recently did not realize that carrier systems themselves may impose risks to the patient The kind of hazards that are introduced by using nanocarriers for drug delivery are beyond that posed by conventional hazards imposed by chemicals in classical delivery matrices For nanocarriers the knowledge on particle toxicity as obtained in inhalation toxicity shows the way how to investigate the potential hazards of nanocarriers The toxicology of particulate matter differs from toxicology of substances as the composing chemical(s) may or may not be soluble in biological matrices, thus influencing greatly the potential exposure of various internal organs

Appropriately engineered nano-sized delivery systems can achieve finer temporal control over drug release rates due to their large surface area Nanocarriers can also be inherently useful in systems that require a burst release Nanocarriers, unlike bulk drug delivery systems, can enter cells to deliver drugs and can be designed to respond to intracellular cues Further, since nanocarriers can circulate in the body after being injected they have the ability to target diseases at the site of disorder This feature of nanocarriers is especially useful in cancer therapy, where the size of the delivery system

is the key to target cancers through the enhanced permeability and retention effect (EPR)

6

Diseased cells can also be targeted by attaching ligands or antibodies to the surface of nano-drug delivery systems Targeting allows nanocarriers to hone into diseased cells by targeting specific features of a disease phenotype, such as an over expressed protein or enzyme Another important aspect of delivery that is now being given the importance it deserves is the drug encapsulation stability in these carriers This is especially relevant because it is increasingly realized that the thermodynamic parameters like percent (%) loading do not adequately describe how stable the delivery vehicle would be during circulation in blood, since these vehicles could potentially leak out drugs into hydrophobic sites in surrounding tissue and blood components Delivery vehicles, based

on a single platform, which can satisfy all basic requirements of a versatile nanoscopic delivery vehicle, are quite rare These features however are the foundations of a good delivery vehicle and are fundamental design requirements Thus there are key aspects of a delivery vehicle design that was described as the basic anatomy of a drug delivery vehicle

3 Self-assembled nanocarrier for drug delivery

Nanoparticles are now available that are attractive for a wide range of materials and devices, but novel fabrication methods are also required to take full advantage of the interesting properties of nanoparticulates Approaches based on the self-assembly of systems from individual components offer tremendous cost advantages and an almost a magical "ease of manufacture" compared to lithographic methods Self-assembled nanoparticles also give the great opportunity in terms of diversity and functionality in the design for defined drug delivery purposes Self-assembled nanoparticles have many

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advantages as highly efficient drug delivery vehicles including nanoscale size, controlled composition and capacity to encapsulate a wide range of drug molecules In particular, by using advanced chemistry and precision engineering at a molecular level, these synthetic polymers provide a wide opportunity for functionalization and versatility which impact the physico-chemical properties of self-assembled systems Examples of self-assembled nanocarriers for targeted drug delivery are showed in Figure 1.2

8

Figure 1.2 Example of self-assembled nanocarriers for targeted drug delivery: a Micelles, an aggregate of surfactant molecules dispersed in a liquid colloid where drugs are physically encapsulated in the inner core b Liposomes, a spherically arranged bilayer structure with drug loaded either in the inner aqueous phase or between the lipid bilayers c Oil/water emulsion, a mixture of liquids that are normally immiscible with drug loaded in the inner oil phase d Nanocapsules, a polymeric membrane which encapsulates an inner liquid core e Nanogels, a nanoparticle composed of a hydrogel f Core-shell particles, the location of nanocrystals at the core with the polymers on the outer layer

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4 PEGylated nanocarriers for systemic delivery

Clearly, particles with longer circulation times have superior ability to reach the tumor site through passive targeting As opsonization is an integral step in the removal of foreign macromolecules by the RES, many efforts for increasing serum stability and extending circulation time have focused on blocking absorption of opsonins onto the nanoparticle surface.5 For passive targeting to be successful, the nanocarriers need to circulate in the blood for extended times so that there will be multiple possibilities for the nanocarriers to pass by the target site Nanoparticulates usually have short circulation half-lives due to natural defense mechanisms of the body to eliminate them after opsonization by the mononuclear phagocytic system (MPS, also known as reticuloendothelial system Therefore, the particle needs to be extended circulation half-lives

Cellular entrapment in macrophages can be avoided by surface modification of the nanocarriers Among many materials used to make or modify pharmaceutical carriers (lipids, natural and synthetic polymers, emulsions, or dendrimers) special attention was paid to polyethylene glycol (PEG, also known as polyethylene oxide (PEO)), which was used both for chemical modification of various drugs (peptide and protein, first of all) to make them more stable and long-circulating and for the decoration of pharmaceutical carriers to improve their pharmacokinetic properties Figure 1.6 illustrates how opsonin proteins associate with foreign bodies and coat its surface As bacteria and viruses have the same negative surface charge as phagocytic cells, opsonins are critical to reducing the charge repulsion between the two systems Next, phagocytic cells engulf the material and

10

transport it to the liver or spleen for degradation and excretion (Figure 1.3 a3–a4) Additional phagocytic macrophages are permanently located in the liver Known as Kupffer cells, these cells serve as a major filter for many types of NPs and are a major interference with long t½ The PEG polymer on a NP surface increases t½ by reducing this opsonization process (Figure 1.3 b2), thus preventing recognition by monocytes and macrophages, allowing the NPs to remain in the blood pool Hydrophobic particles are also more vulnerable to the RES and hydrophilic PEG reduces these complications In addition to NP–RES interactions, poor t½ can also result from NP–NP interactions (i.e., aggregation) NPs aggregate primarily because the attraction between particles is stronger than the attraction for solvent For spherical NPs, the interaction potential is related to the electrostatic repulsive potential and the van der Waals attraction potential PEG decreases the surface energy of NPs and minimizes van der Waals attraction.6

Prior to NP applications, PEG was used as a nontoxic, water-soluble dispersant/stabilizer FDA approved PEG is a highly hydrophilic, flexible polymer which has an inherent long circulating property The array of already available versatile PEG chemistries make it an attractive polymer to be used in modifying pharmaceuticals or surfaces of pharmaceutical carriers to achieve the desired long-circulating property or add convenient functional groups to conjugate ligands for active targeting Early work with PEGylated NPs stemmed mostly from drug delivery.7 One of the first reports on PEGylation was described by Davis and Abuchowski,8 where they covalently attached methoxy-PEGs (mPEGs) of 1900 and 5000 Da to bovine serum albumin and to liver catalase Later, acrylic microspheres functionalized with PEG-modified human serum albumin increased t½ in vivo.9 Li and colleagues found that 75-nm latex particles

11

remained in rat circulation 40-times longer (half-life 20 min vs 13 h) when coated than uncoated with PEG larger than 5000 kDa.7b In the mid-1990s, Doxil® (liposomal delivery vehicle for doxorubicin) and oncospar (PEG-l-asparaginase) became the first FDA-approved NP therapeutics.7c Doxil increases doxorubicin bioavailability nearly 90-fold at 1 week from injection of PEGylated liposomes versus free drug.10 Later, Abraxane® was introduced as an albumin-functionalized NP for delivery of taxane without cremphor to enhance drug efficiency.11

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5 Targeted drug delivery systems for cancer therapy

Conventional cancer chemotherapies have dose-related side effects owing to nonspecific biodistribution of drugs Targeted nanomedicines are emerging as one of the promising approaches in anticancer treatment and have major advantages Targeting active molecules to specific sites in the body had been pursued actively ever since Ehrlich first envisaged the use of 'magic bullets' for the therapy of various diseases.13 Interest in this concept has increased significantly in recent decades with the innovations of nanomedicine Cancer nanomedicines have the ability to improve the therapeutic index of drugs by preferential localization at target sites, lower distribution in healthy tissues, delivery of hydrophobic drugs and extended release rate Progress in the development of nanomedicines for targeted drug delivery has been reviewed by Moghimi and colleagues.14 Targeted delivery can be achieved passive, active targeting, or their combinative targeting

14

Figure 1.4 Conceptual representation of nanoparticle tumor-targeting modalities Passive targeting: Unlike that found in normal tissue, tumor vasculature is leaky owing to fenestrations and gaps between endothelial cells that result from abnormal angiogenesis NPs in circulation can passively extravasate through these gaps and enter the tumor interstitium Poor lymphatic drainage found in some tissues helps to retain particles in the tumor space Active targeting: Ligands (e.g antibodies, peptides, small molecules, etc.) targeted toward moieties overexpressed or uniquely present on the plasma membrane of tumor cells can be used to actively enhance NP accumulation at the tumor site and can also help to internalize particles into cells via endocytosis.15

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5.1 Passive targeting strategies and recent developments

Cellular barriers present formidable obstacles in the delivery of therapeutics for cancer treatment Fortunately, certain aspects of cancer physiology can be exploited to achieve passive targeting to tumor sites Rapid growth of tumors leads to aberrant angiogenic vasculature The newly formed blood vessels are often disorganized and discontinuous, resulting in increased permeability to macromolecules Moreover, lymphatic drainage systems are often poorly developed or non-existent in tumor sites, enabling accumulation of therapeutics.16 This phenomenon, called the enhanced permeation and retention (EPR) effect has increased the tumor concentration of anticancer agents up to 70-fold in some cases.17 Since the pioneering work of Couvreur et al.,18 nanoscale systems have been aggressively investigated for their utility in drug delivery applications (Figure 1.4)

Nanoparticle size is known to play a critical role in achieving passive targeting The majority of solid tumors exhibit a vascular pore cutoff size between 380 and 780 nm.19Therefore, particles need to be of a size much smaller than the cutoff pore diameter to reach to the target tumor sites By contrast, normal vasculature is impermeable to drug-associated carriers larger than 2 to 4 nm compared to free, unassociated drug molecules.20

This nanosize window offers the opportunity to increase drug accumulation and local concentration in target sites such as tumor or inflamed sites by extravasation, and significantly to reduce drug distribution and toxicity to normal tissues Nanocarriers above 10 nm in diameter are generally able to avoid filtration by the kidneys, while less well understood, the upper size limit for passively targeted nanocarriers is thought to be

16

approximately 150 nm.21 Extravasation and diffusional barriers limit nanoparticle access

to tumors when particle size is over 200 nm.21b Additionally, previous studies have shown that nanoparticle clearance rate increases with size.22 One such investigation demonstrated that the blood clearance of 80 nm nanocarriers was half as fast as the clearance of 170 and 240 nm particles Presumably, this effect is due to non-specific protein adsorption on the surface of larger nanocarriers, leading to opsonization and subsequent clearance by the RES.22

5.2 Active targeting strategies and stimuli-triggered ligand presentation

Localized diseases such as cancer or inflammation not only have leaky vasculature but also overexpress some epitopes or receptors that can be used as targets Therefore, nanomedicines can also be actively targeted to these sites Ligands that specifically bind

to surface epitopes or receptors, preferentially overexpressed at target sites, have been coupled to the surface of long circulating nanocarriers.23 Ligand-mediated active binding

to sites and cellular uptake are particularly valuable to therapeutics that are not taken up easily by cells and require facilitation by fusion, endocytosis, or other processes to access their cellular active sites.24 Active targeting can also enhance the distribution of nanomedicine within the tumor interstitium More recently, active targeting has been explored to deliver drugs into resistant cancer cells.25 An important consideration when selecting the type of targeting ligand is its immunogenicity For example, whole antibodies that expose their constant regions on the liposomal surface are more susceptible to Fc-receptor-mediated phagocytosis by the mononuclear phagocytic system.26 Examples of targeting ligands and their targets are listed in Table 1.1

17

Table 1.1 Selected examples of ligands used in active drug targeting

and therapy Non-peptidic RGD

mimetic

avβ3 integrin Integrin positive cell

imaging Mimetic of the sialyl

domain

Phospholipids Apoptosis imaging

disease imaging EPPT1 (YCAREPPT

RTFAYWG)

Underglycosylated mucin-1 antigen

Multiple tumor type imaging

Aptamers A10 RNA aptamer Prostate-specific

membrane antigen

Prostate cancer imaging Thrm-A and Thrm-B

DNA aptamers

Human thrombin protein

alpha-Serum protein detection Proteins Annexin V Phosphatidylserine Apoptosis imaging

imaging Transferrin Transferrin receptor Breast cancer

imaging Antibodies Monoclonal antibody

A7

Colorectal carcinoma Colon cancer

imaging Herceptin

(Trastuzumab)

Her2/neu (Breast cancer)

Breast cancer imaging and therapy Rituxan (Rituximab) CD20 antigen Lymphoma imaging

therapy RGD, Arg-Gly-Asp tripeptide: LHRP, luteinizing hormone releasing hormone; Endothelial vascular adhesion molecule-1, VCAM-1

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6 Stimuli-response for controlled drug delivery

6.1 Concepts for designing stimuli-responsive nanocarriers

Despite the fact that stability of encapsulation in a delivery carrier is necessary during circulation, drug delivery will only be effective if the drug is released once it reaches its intended target Releasing specific sites in the body simplifies drug administration procedures, reduces the quantity of drug required to reach therapeutic levels, decreases the drug concentration at on-target sites (possibly reducing side effects) and, essentially, increases the concentration of the drug at target sites This can be reached by incorporating chemical moieties into the design that make the carrier responsive to stimuli relevant to the disease being targeted

Interest in stimuli-response is steadily gaining increasing momentum especially in the fields of controlled and self-regulated drug delivery Delivery systems based on stimuli-response are developed to closely resemble the normal physiological process of the diseased state ensuring optimum drug release according to the physiological need There are two kinds of stimuli, broadly defined, that can be engineered into delivery systems:

internal stimuli (i.e., enzymatic reactions, changes in pH, redox, and temperature) and external stimuli (i.e., heat, light, magnetic and electrical fields) When drug delivery systems maintain a response interaction they necessarily require a stimuli-response to cleave the interaction The release of drug is triggered by the stimulus (Figure 1.5)

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Figure 1.5 Dual and multi-stimuli responsive polymeric nanocarriers as emerging controlled drug release systems There are two kinds of stimuli, broadly defined, that can

be engineered into delivery systems: internal stimuli (i.e., enzymatic reactions, changes in

pH, redox, and temperature) and external stimuli (i.e., heat, light, magnetic and electrical fields)

20

6.1.1 Internal stimuli

Internal stimuli of chemical and biochemical origin include cellular pH-shift, redox, and ionic microenvironment of the specific tissues, enzyme over-expression in certain pathological states, host–guest recognitions, and antigen–antibody interactions (Figure 1.6).28

pH stimulus

In the pathological state, the normal pH-gradient existing between extra and intracellular environment is greatly affected A well-established fact is that in solid tumors, the extracellular pH can be significantly more acidic (~ 6–7) than systemic pH (7.4) due to poor vasculature and consequent anaerobic conditions prevailing in the malignant cells.29 Besides, the cellular organelles also exhibit sharp pH differences in different locations, for instance, in cytosolic, endosomal, and lysosomal compartments A polymeric nanocarrier with pH-sensitive modality can register such pH-gradients and, as

a response, can facilitate the release of the payload near the target compartment either by destabilization of the nanocarrier itself or by decomposition of the pH-sensitive linking unit that connects the drug to the carrier A number of nanocarrier-mediated gene transfer approaches have already been extensively studied where the destabilization of the internalized nanocarriers are brought about via a “cross-talk” of nanocarrier surface-charge and environmental pH condition, resulting in the release of the genetic materials

to the cells.30

Redox stimulus

Enzyme stimulus

A number of biochemical signatures, which are specific for a diseased tissue, can also

be used as triggering factors for drug release from SRNs An intracellular protease cathepsin, especially cathepsin B that degrades proteins in lysosomes, has been heavily investigated for the development of enzyme-responsive nanocarriers Generally, the proteases that are extracellular expressed, such as the matrix metalloproteases, are specific biomarkers of malignant tissues and are responsible for the proteolysis of the extracellular matrix and basement membranes and are required during embryo morphogenesis, tissue remodeling, angiogenesis, and parasitic or bacterial invasion.33These biochemical signatures can act as a trigger when spatially oriented drug release is required This can be achieved by introducing specific enzyme substrate sequence either into the nanocarrier scaffold, or in the linker segment through which the drug is anchored

on to the nanocarrier

22

Thermo stimulus

Increases in temperature are associated with several disease states (e.g., cancer 34) Thermo-responsive drug carriers have been employed to release their payload within environments above the physiological temperature Thermo-sensitive polymers exhibit a phase transition in solution at a temperature known as the lower critical solution temperature (LCST) For example, PNIPAm, a well-studied thermo-responsive polymer, undergoes a reversible phase transition in aqueous solution from hydrophilic to hydrophobic at its LCST of approximately 32°C Chemical modifications of PNIPAm have been effective in controlling the LCST.35 In 2005, Liu et al synthesized poly(N-isopropylacrylamide-co-N,N-dimethylacrylamide)-b-poly(D,L-lactide-co-glycolide) micelles for controlled paclitaxel delivery.36 Paclitaxel release was accelerated when the physiological temperature was raised above the LCST The paclitaxel-loaded micelles were more effective in killing human breast carcinoma cells at 39.5°C than 37°C De and colleagues developed folate-conjugated, thermo-responsive block copolymer micelles Folate is known to bind to several cancer cell types.37 The drug release studies from folate-conjugated PNIPAm-DMA micelles demonstrated a temperature-responsive drug release Delivery of paclitaxel at the tumor site can alter the overall drug biodistribution Needham et al developed temperature-sensitive liposomes containing doxorubicin.34aTheir liposome formulation, composed of 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine (MPPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), hydrogenated soy sn-glycero-3-phosphocholine (HSPC), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-polyethylene glycol 2000 (DSPE-PEG-2000), was optimized to

23 rapidly release the drug under mild hyperthermic temperatures (39 °C to 40 °C) Changing the drug biodistribution can increase therapeutic efficacy

24

Figure 1.6Schematic illustration of block copolymer assemblies which can respond to a range of stimuli characteristic of tumor tissues and intracellular microenvironments, promoting targeted delivery and controlled release of therapeutic drugs and imaging agents.38

25

6.1.2 External stimuli

Physical stimuli that can be applied externally to bring about a triggered release of active guest may involve temperature, light, mechanical pressure, and strength of magnetic or electrical fields.39

Thermo responsive

With the onset of advanced research in the field of hyperthermia and their purported advantage in increasing vascular perfusion and permeability, thermo-responsive polymers have become an attractive candidate for designing therapeutic nano-vehicle for target specific delivery of bioactive agents It should be mentioned that, the temperature range within which the thermo-responsive nanocarriers should release their cargo falls between

37 and 42 °C, as above this temperature protein denaturation and disruption of fine anatomical structures are imminent Feasibility of local/regional heat deposition and hyperthermia induced vascular permeability additionally endow the thermo-responsive nanocarrier the advantage of remote targeting in passive mode.28, 31a

Light stimulus

Utilization of light as an external stimulus offers a range of advantages, including ease of application, relative biocompatibility and controllability both spatially and temporally.40 The principle of photo-responsive dendritic architectures relies on the adjustable release of encapsulated/conjugated bioactive units from the structure under the influence of light of specific frequency.28 In particular, radiation of UV, near IR, and IR frequency are generally used which are tissue compatible, yet powerful enough to bring about conformational changes within the nanocarriers' chemical architecture

6.2 Previous studies of stimuli-response for controlled drug delivery

These stimuli-responsive polymeric nanocarriers have demonstrated improved drug release behavior and anti-tumor activity to varying degrees, depending on type of stimulus, rate of response, and exact spot of triggering drug release In an effort to further fine-tune drug release and augment therapeutic efficacy of nano-particulate drugs, sophisticated polymeric nanocarriers that respond to dual and multi-stimuli such as pH/temperature, pH/redox, pH/magnetic field, temperature/reduction, double pH, pH and diols, temperature/magneticfield, temperature/enzyme, temperature/pH/ redox, temperature/pH/magnetic, pH/redox/magnetic, temperature/redox/ guest molecules, and temperature/pH/guest molecules have been aggressively pursued It should be noted that the responses take place either simultaneously at the same location or in a sequential manner in different settings and/or compartments These dual and multi-stimuli responsive polymeric nanocarriers might on one hand offer unprecedented control over drug delivery and release leading to superior in vitro and/or in vivo anti-cancer potency,

27

and on the other hand also facilitate nanoparticle preparation and loading of drugs under mild conditions

For example, redox-sensitive drug release polymersomes have been developed based

on temperature and reduction dual-responsive PEG-PAA-PNIPAAm triblock copolymers

by simply increasing solution temperature to above their lower critical solution temperature (LCST) followed by crosslinking with cystamine via carbodiimide chemistry.24,25 These cross-linked polymer-some while robust against physiological conditions were rapidly dissociated to release exogenous proteins in cancer cells due to redox-triggered de-crosslinking and disruption of polymersomes pH and redox dual-sensitive disulfide-crosslinked micelles were developed to reduce premature drug release

in blood circulation, enhance drug accumulation in the tumor site, and actively release drug in the target tumor cells in response to endo/lysosomal pH and intracellular reducing environment.26

In another study multiple DOX molecules were chemically conjugated via cleavable hydrazone linkages to the repeating units of PPO chains of a Pluronic-mimicking triblock copolymer The DOX-copolymer conjugates spontaneously formed polymeric micelles The conjugated DOX was released from the micelles due to the cleavage of hydrazone linkages, which was accelerated at acidic pH 5 compared to pH 7.4 DOX-copolymer conjugate exhibited a different intracellular distribution profile and enhanced cytotoxicity with respect to MCF7 cells compared to free DOX It was suggested that DOX-copolymer conjugate transported into cells via endocytosis as opposed to transmembrane diffusion realized in the case of free DOX.42 A pH-dependent

pH-28

non-covalent incorporation and release of DOX in Pluronic P85-poly(acrylic acid) block copolymer was reported.43 In this case at the extracellular pH the DOX molecules were apparently bound to the micelles due combination of hydrophobic interactions with PPO chains and electrostatic interactions with carboxylic groups of poly(acrylic acid) Acidification at pH 5.0 resulted in protonation of the carboxylic groups and release of the drug

7 Overall objectives

A wide range of contents extending from carcinogenesis to current methodologies for targeted drug delivery are described in brief These all categories provide a promising way toward the design of nanocarriers for targeted drug delivery Recently, research on programmable self-assembly of nanosystems promises to bring a new paradigm in relation to the fabrication of materials and devices Although significant progress is evident, several major challenges still need to be understood and resolved In this area, one of the most desirable approaches for enhanced therapeutic efficacy is the development of nanocarriers that can meet the following requirements: (i) maintaining high structural stability in blood, (ii) eliminating undesirable drug release before reaching the target site, and (iii) releasing drugs specifically within target cells The overall objectives of this dissertation are as follows;

1) To prepare and characterize of self-assembled nanogels based on reducible heparin-Pluronic copolymer, pH- and redox-stimuli sensitive Pluronic micelle, and self-assembled magnetic nanocarriers based on host-guest inclusion for anticancer target delivery

29

2) To evaluate these nanocarriers toward; the general features of nanocarriers, response of nanocarriers to pH, redox potential, or extra molecule stimuli in triggered drug release, and ability of c(RGDfC) and folic acid targeting ligand to promote the nanocarriers into HeLa cells

3) To test in vivo therapeutic efficacy of the nanocarriers on breast cancer 7/ADR)

(MCF-30

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World Health Organ 89: 716-24, 24A-24C

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