Accepted Manuscript

A review of drug release mechanisms from nanocarrier systems

Chizhu Ding, Zibiao Li

PII: S0928-4931(17)30728-2
DOI: doi: 10.1016/j.msec.2017.03.130
Reference: MSC 7641
To appear in: Materials Science & Engineering C
Received date: 23 February 2017
Revised date: 15 March 2017
Accepted date: 17 March 2017

Please cite this article as: Chizhu Ding, Zibiao Li , A review of drug release mechanisms
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 ACCEPTED MANUSCRIPT

A review of drug release mechanisms from nanocarrier systems

Chizhu Dinga, Zibiao Lib,*

a
College of Science, Huazhong Agricultural University, Wuhan, China
b
Institute of Materials Research and Engineering, A*STAR (Agency for Science,

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Technology and Research); 2 Fusionopolis Way, Innovis, #08-03, Singapore 138634,

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Singapore.

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Corresponding author: Z. Li (lizb@imre.a-star.edu.sg) Tel: +65-63194767
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Abstract

The most common methods used for drug administrations are pills, injections, lotions and
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suppositories. The preferred means is oral dosage forms as it is simple, painless and self-

administered. However, the drugs are usually degraded within gastrointestinal tract or not
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absorbed in sufficient quality to be effective. Over the years, a variety of other administration
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means have evolved to show specific advantages for particular agents and certain diseases. In
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this review, various nano-delivery systems consisting of different covalent linkages to

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conjugate the therapeutic molecules as well as those that carry the unmodified drug

molecules by encapsulating or complexation are summarized, including ester, amide/peptide,

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disulfide, hydrazone, hypoxia-activated and self-immolative linkages. The mechanisms for

controlled drug release are also discussed. In addition, the new mechanism of the recently

developed photochemistry or thermolysis to trigger controlled drug release and the

applications are also covered.

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Keywords: Drug delivery; Controlled release; Nanocarrier; Prodrugs; Reliable linkers;

Cancer therapy;

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1. Introduction

More than four thousand years ago, Egyptian physicians started using drugs in the form of
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pills, ointments and salves as treatments for illnesses. The first intravenous injection was

done in human in 1665 and subcutaneous injections were introduced in 1853.[1]

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Subsequently, the modern hypodermic syringe was developed in 1884. The current means of
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drug administration still have close resemblance with these ancient methods and undergo
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little change throughout the years. Some of these administration means developed over the
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years have specific advantages for particular agents or certain diseases.[2] Table 1 shows a

brief summary of the common routes of drug administration.[3]

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Research and developments in creating new and innovative drug delivery systems are

increasing at a fast pace globally due to the increasing demands in low cost and higher

efficiency for better therapies.[4] To meet this demand, many well-known and effective

applied drugs will be reformulated in new drug delivery system to provide enhanced

efficiency or more beneficial therapy. One of the most promising drug delivery systems is

nanotherapeutic delivery system.[5] It is a drug delivery concept in nanoscience and it is

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often refers to as an strategy that develops platforms and nanoscales devices for selective

delivery of therapeutic genes as well as small drug molecules to the cells of interest.[6] In this

paper, the release mechanisms of the drug in nanotherapeutic delivery systems will be

covered based on the linker types for the various drugs that are conjugated to nanocarriers. A

discussion on how the mechanisms are designed and used in the delivery platform to provide

specific targeted release to specified cells will also be mentioned.

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Administration routes Examples Pros Cons

Intravenous injection Antibiotics for sepsis 100% bioavailability Discomfort to patient, Requires health
care provider, Risk of overdose or
toxicity, Risk of infection
Intravenous infusion Heparin for anticoagulation 100% bioavailability, Continuous Requires hospitalization, Risk of

Subcutaneous injection
control over plasma levels
Usually high bioavailability
P T
infection
Discomfort to patient
Intramuscular injection
Oral
Insulin for diabetes
Aspirin, acetaminophen,
Usually high bioavailability
Convenient, Self-administered R I
Discomfort to patient
Drug degradation prior to absorption,
Sublingual or buccal
ibuprofen
Nitroglycerin for angina
S C
Avoids first-pass metabolism in liver,
Self-administered
Limited absorption of many drugs
Limited to lipophilic highly potent agents

Ophthalmic Pilocarpine for glaucoma

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Local delivery, Self-administered
N
Discomfort to some patients, Frequent
administration
Topical
Intra-arterial injection
Antibiotic ointments
Chemotherapy, in some
cases
A
Local delivery, Self-administered
Control of vascular delivery to specific
regions
M
Limited to agents that are locally active
High risk

Intrathecal injection Pain medication, in some Direct delivery to brain Limited drug penetration into brain

Rectal
cases

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Avoids first-pass metabolism in liver,
tissue, High risk
Discomfort leads to poor compliance in

P T Self-administered some patients

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Transdermal Nitroglycerin patches for Continuous, constant delivery, Self- Skin irritation, Limited to lipophilic,
angina administered highly potent agents
Vaginal

C C
Spermicides Self-administered Discomfort leads to poor compliance in
some patients
Controlled release of
implants
A
Norplant for contraception Long-term release Requires surgical procedure

Table 1. The common routes of drug administration. Reproduced from ref [3] with permission.

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1.1. Nanocarriers based therapeutic delivery

There are infinite possibilities to the application of nanotechnology in current drug delivery

systems. Most biological functions are highly dependent on nanoscale dimension units like

viruses, ribosomes and molecular motors.[7] Thus by having nanoparticles that are small

enough for direct interaction with subcellular compartments, it opens up the possibility of

activating intracellular events.[8]

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In general, the nanocarriers based therapeutic drug delivery systems are preferred over

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normal drug delivery due to the numeral promising advantages.[9] Firstly, the larger surface-

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to-volume ratio of the nanocarriers could allow a larger contact area of the drug with the body

in the same drug concentration condition, thus the same dose of drugs makes the delivery
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more effective.[10] The reduced dosage will in turn reduce the drug's side effects and toxicity

issues. Secondly, nanocarriers have tuneable surface chemistry for different drugs and
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targeting molecules. For example, the highly efficient drug doxorubicin (DOX), which is

used to treat various types of tumours also has a major side effect of causing serious
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cardiotoxicity due to the lack of tumour specific cytotoxicity. The tuneable surface chemistry
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allows the possibility of having prolonged and sustained drug release, this improves the
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bioavailability of the drug to where and when it is most needed and also allows longer period
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of drug circulation than the drug alone.[7] For instance, one can make drugs that are

hydrophobic and has low aqueous solubility to be transported in vivo conditions. The surface
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chemistry modification allows efficient navigation in the complex in vivo environment by

protecting the drug from undue degradation. The surface chemistry modification also enables

not only the drug to be directed to specific cell types for targeted delivery but even to special

regions of the cell making enhanced intracellular trafficking of the drug possible.[11] Lastly,

the nanocarriers can provide the flexibility in the forms of having more diverse routes of drug

administration and also in terms of drug formulation.

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The nanocarriers that carry the drugs can be made in different forms like dendrimer, liposome,

rods and many other forms. These nanocarriers can be organic based dendrimer nanoparticles

(NPs),[12] polymer,[13] polymer based micelles,[14] hydrogels[15, 16] or inorganic based

like gold nanoparticles(AuNPs), magnetic nanoparticles,[17] semiconductor

nanoparticles,[18] carbon nanotubes(CNTs) or even ceramics like iron oxide nanoparticles

(IONPs).[19] The different forms of nanocarriers are as shown in Figure 1.[20] They are

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versatile and often have many different functions. Normally the therapeutic agents are

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dissolved, adsorbed, entrapped, encapsulated or attached on the surface or inside the

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nanocarriers. These nanocarriers are currently used as targeted nanotherapeutics and also in

diagnostic test for inflammatory, infectious and autoimmune diseases as well as cancer.[21-
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23] When the nanocarriers are in the blood streams, the plasma proteins, cells and other blood

components will interact extensively with the material, some of the interactions aids the
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transportation of the nanocarriers with the drugs to the targeted sites.[24]

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Figure 1. Schematic illustration showing the different types of nanocarriers used in drug

delivery. Reproduced from ref [20] with permission.

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The nanocarriers can be modified to have its own chemical and physical properties by

altering the synthesis method, surface functionality and modification, size, shape and the bulk

structure.[25] To develop an effective therapeutic delivery system, one needs to know the

physicochemical properties of the material and how it interacts with the biological systems in

our body.[26] For instance, optimal surface modifications are required to eliminate or reduce

undesirable effects like intrinsic toxicity and immunogenicity, which some of the

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nanomaterials may have. Using cationic nanoparticles as an example, they are cytotoxic due

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to its nature to disrupt cellular membranes, but by modifying the surface with neutral or

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anionic groups we can reduce or eliminate the cytotoxicity.[13, 27] By doing surface

modification, many of the inorganic materials like cobalt-chromium nanoparticles which are
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originally toxic in the body can be used. Similarly, the properties of the nanocarriers are

custom made for the effective drug loading and efficient mechanisms for release of the
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therapeutic agent.[28]

The main function of these nanocarriers is to transport the therapeutic drugs to the targeted
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site. The process involved in the cellular delivery of therapeutic agents usually involved
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passive diffusion, particle phagocytosis, pinocytosis or receptor-mediated endocytosis.[3]

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This is done by either passive targeting approach or active targeting approach.[29] Figure 2
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shows the schematic illustration of drug carrying nanoparticles in tumour directed delivery

based on the active and passive targeting. Passive targeting makes use of increase in
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endothelial blood microvasculature permeability in tumours due to the larger interstitial gaps

between the neighbouring cells to facilitate the delivery of the drug loaded nanocarriers into

the tumours.[30] This enhanced permeation and retention (EPR) effect allows greater

accumulation of drugs as well as longer drug exposure duration in the tumour due to limited

elimination.[31] Active targeting approaches make use of EPR effect and the targeting

ligands that are covalently bonded to the nanocarriers surface to target on specific cancer

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cells. These targeting ligands are specific for a particular cell surface biomarker or over-

expressed receptor molecules in the cancerous cells.[20, 30]

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Figure 2. Schematic illustration of drug carrying nanocarriers in (A) passive targeting and (B)

active targeting in tumor-directed delivery. Ligands attached to the surface of nanocarriers

bind to receptors over/expressed by cancer cells or endothelial cells. Reproduced from ref [29]

with permission.

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The common surface biomarkers that are overexpressed in cancers and inflammatory diseases

can be classified under vitamin receptors, αvβ3 integrin receptor, PSMA (prostate-specific

membrane antigen) receptor, growth factor receptor, insulin and insulin-like receptors,

selecting protein molecules and transferrin.[20] Under the vitamin receptors family are folic

acid (FA, vitamin B9) receptors (Far-α, FAR-β), riboflavin (vitamin B2) receptor and biotin

receptor and under the growth factor receptors family are fibroblast growth factor receptor

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(FGFR), Her2 as well as epidermal growth factor receptor (EGFR). The targeting ligand is

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usually attached in multiple copies to the nanocarrier surface since the mechanism for

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endocytic uptake of these targeted carriers requires the combined occurrences of several

concurrent interactions at the contact point of many pairs of surface receptor and ligand.[32]
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As a result of the multivalent binding mechanism, tight adhesion between the nanocarriers

and targeted cell surface is obtained during the receptor-mediated uptake of the nanocarriers
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by the targeted cell.[25, 33]

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2. Drug release mechanisms through linker cleavage

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This paper covers on the nanocarrier systems that use different covalent links to attach and
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carry the therapeutic molecules as well as those that carry the unmodified drug molecules by
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encapsulation or complexation. The importance of the linkers that serves as a covalent

attachment for carrying the therapeutic molecules as well as how the linkers are used to
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control the release mechanism of the drug are highlighted. The insights on the drug release

mechanisms will be discussed based on the structure and functions of the different types of

linkers. In addition, the recently developed mechanisms of using photochemistry or

thermolysis to trigger drug release in an actively controlled manner and their applications will

also be discussed.

2.1. Cleavage by hydrolysis

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2.1.1. Hydrolysis of ester bond

Carboxylic ester based linkers are used in the targeted drug release mainly due to the wide

use of ester linker to attached to many therapeutic molecules and convenience. The

conjugation or ester based drug attachment is formed between the nanocarrier and drug by

having a pair of functional groups like carboxylic acid or alcohol.[34] The ester bond formed

allows the drug release to take place due to its susceptibility to the hydrolysis process in vivo

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under physiological conditions. This hydrolytic reaction is catalysed by the presence of acids,

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bases, metal ions like copper(II) ions and hydrolytic proteins like esterases and human serum

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albumin. Owing to the physiological instability of ester bond in vivo, it is not effective and

specific enough to allow controlled drug release to take place.

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One of the main process of drug delivery is receptor-mediated endocytosis where the

nanocarriers are engulfed into the cytosol through coated pits and become part of the
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endosome before it is being fused with lysosomes.[35] During the process of endocytosis,

numerous acid hydrolases like cholesteryl ester acid hydrolases, aryl sulfatase, acid
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phosphatase, N-acetylglucosaminidase and cathepsin D are present.[36] These hydrolases

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will aid the ester or amide hydrolysis including ester based linkers between the drug and
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nanocarriers. More optimal catalytic activity will take place causing drug release to occur
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more rapidly and selectively after uptake if the environment caused by the endosomes is

between pH 5.0 to 6.0 and lysosomes is at pH 4.8 instead of the neutral environments like
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plasma and cytoplasm with pH 7.4.[37] In short, the mechanism of cleaving of ester linkers

that causes drug release is strongly connected to the specific uptake by the targeted cell and

the intracellular linker hydrolysis which is catalysed by the acid hydrolases in the endosomes

and lysosomes.[36] There are other hydrolytic enzymes that associated with the disease and

overexpressed in the plasma or extracellular membrane. Due to the abnormalities in diseased

cells under pathological conditions, it can cause upregulation in expression producing

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enzymes that catalyse the hydrolysis of the ester linkers causing drug release to take place.

For instance, a Zn2+/Mg2+ dependent membrane-bound metalloenzyme, alkaline phosphatase

is proven to release a quinolinone drug molecule by acting as catalyst for the hydrolysis of

the drug to O-phosphate bond.[38] In addition, a serine-dependent esterase, Carboxylesterase

2 (CE-2) found on the membrane comprises one of the biomarkers proteins that is expressed

in abundances in liver and colon tumour. It will catalyse the hydrolysis of endogenous lipid

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esters and also cleavage of carbamate-based prodrug substrates like irinotecan (CPT- 11), an

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anticancer agent causing drug release.[39] The cleavage of CPT- 11 releases SN38 as an

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active metabolite which inhibits topoisomerase I.[39]

On the other hand, the chemical mechanism like hydrolysis reaction will also cause the ester
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linkers to be cleaved and is catalysed by the presence of acid, base and metal ions as

mentioned previously.[40, 41] Most esters are actually more stable in slightly acidic
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condition of pH 5 to 6 as ester hydrolysis through an acid or basic catalyst has the least

impact at this pH range. Thus the drug release caused by chemical hydrolysis of the ester
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linkers that bonds the drug to nanocarriers is insignificant even after the uptake into acidic
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endosomes and lysosomes or on exposure to the extracellular matrix (ECM) of the tumour
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which has a pH of 6.2 to 6.9. This ester linker has high susceptible to base-catalyzed
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hydrolysis which allows controlled drug release to take place in basic subcellular

compartments like mitochondrial matrixes which have a pH of 7.9 to 8.0 and peroxisomes
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which has a pH of 8.2.[42] It was observed that the rate of hydrolysis of ester linkers in the

ester conjugates of paclitaxel is 10 times faster with every one unit increase in pH.[43]

Control of drug release through ester hydrolysis mechanism has been widely exploited in

previous investigations, including Taxol, Methitreate, Platinum agents and SN38. For

example, the taxane class of anticancer agents like paclitaxel and docetaxel are classified as

microtubule-stabilizing agents since they are able to bind and stabilize cytosolic

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microtubules.[44] The microtubules are hollow cylindrical polymers which are made up of

repeating units of heterodimer, α-tubulin and β-tubulin.[45] On the luminal side of the tubular

structure, the taxol molecule will bind selectively to the β-tubulin unit. This stabilization of

cytosolic microtubules inhibits cell growth by impeding with the cellular division process and

the mobility which is highly dependent on the microtubules' assembling and disassembling

ability. An ideal taxol drug delivery system requires a mechanism of drug release after the

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drug conjugate is uptaken by the cells.[46] The taxol are conjugated to the nanocarriers by

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using the secondary alcohol at the C2 position or linked by ester bond at C10 position. An

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example of a clinically certified taxol conjugate is polyglumex (Xyotax) which is made of

biocompatible poly(L-Glu) polymer. Each of the polymer molecules consists of a few

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paclitaxel molecules which are linked by an ester linkage at the C2 position to γ-carboxylic

acid in the polymer.[47] The drug will travel into the cancer cell through endocytosis and gets
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released by lysosomal enzymes like cathepsin B. However, it was also reported that is too

unstable as can be degraded rapidly and release the drug even without enzymes. Other than
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direct chemical hydrolysis and enzymatic hydrolysis of the ester linkage, the release of taxol
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can take place by ester hydrolysis through the addition of disulfide group next to an ester
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linkage.[48, 49] Other types of chemicals used are single walled carbon nanotube (SWNT)
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and triazine dendrimer. For instance, Dai et al. demonstrated the conjugation of PTX to

branched poly(ethylene glycol) (PEG) chains on SWNTs via a cleavable ester bond to obtain
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a water soluble SWNT-paclitaxel conjugate (SWNT-PTX) (Figure 3).[48] The authors noted

that the SWNT-PTX formulation afforded higher efficacy in suppressing tumor growth than

clinical Taxol® in a murine 4T1 breast-cancer model. This is because of the prolonged blood

circulation and 10-fold higher tumor PTX uptake by SWNT delivery. It showed that the drug

molecules carried into the reticuloendothelial system could be released from SWNTs and

further excreted via biliary pathway without causing obvious toxic effects to normal organs,

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indicating promising potential for high treatment efficacy and minimum side effects for

cancer therapy.[48]

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Figure 3. Schematic illustration of paclitaxel conjugation to SWNT functionalized by
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phospholipids with branched-PEG chains. The PTX molecules are reacted with succinic

anhydride (at the circled OH site) to form cleavable ester bonds and linked to the termini of
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branched PEG. This allows for releasing of PTX from nanotubes by ester cleavage in vivo.

Reproduced from ref [48] with permission.

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Methotrexate (MTX) is an important drug under the antifolate molecules family which are
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currently used to treat cancers and inflammatory diseases. When used in subnanomolar

concentration, it shows signs of inhibition of cytosolic dihydrofolate reductase.[50] This

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dihydrofolate reductase acts as a catalyst in the reduction of dihydrofolate to tetrahydrofolate.

The tetrahydrofolate is a cofactor involved in the de novo biosynthesis of thymidine and also

linked to the building block in DNA. However, MTX has a narrow therapeutic index owing

to its dose-limiting systemic toxicity, hence it is more suited for usage in targeted drug

delivery system. [51] There are two carboxylic acids present on the L-Glu portion of each

MTX molecule, they are both able to be used to covalently bond nanocarriers by ester

linkages. Recently, Majoros et al. reported that the possibility of conjugating MTX to

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PAMAM dendrimer fifth generation (G5) by EDC based ester linker. This fifth generation

PAMAM dendrimer was also conjugated to folic acid receptor targeting ligand, FA. In vitro,

the uptake in conjugate through FAR- positive KB cell line undergo receptor-mediated

endocytosis shows possibility of inhibiting cell multiplication (Figure 4).[52] In vivo, the

conjugate demonstrated improvement in therapeutic efficacy in treatments for epithelial

cancer, head, neck tumours and inflammatory arthritis. However, the exact mechanisms of

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action and release for MTX conjugated by ester linkage is yet to be known. It is important to

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highlight that in acidic conditions that is similar to the intracellular environment of

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endosomes, the ester conjugate of MTX displayed great resistance against the hydrolysis

process which may affect the cellular uptake. Other MTX delivery systems that make use of
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ester linkages are glycidylated PAMAM dendrimer which are FA conjugated, MTX

conjugated to G3 PAMAM dendrimer, MTX prepared by "one pot" reaction, MTX

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conjugated to G5 PAMAM dendrimer by a triazine spacer through azide-alkyne click

chemistry.[53, 54]
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Figure 4. The diagram showing the conjugation of MTX to G5 PAMAM dendrimer by ester

linkage and its anti-tumour mechanisms. Reproduced from ref [52] with permission.

In another aspect, platinum agents are among the most powerful and widely used

chemotherapy drugs against cancer. The first chemical of the platinum-based anticancer

therapeutic agent family discovered is cisplantin, Pt(II) (NH3)2Cl2. The two amine ligands in

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cisplantin are strongly chelated to the platinum ion and thus they have low reactivity towards

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carboxylic acid to form amide linkage.[55] Cisplantin conjugation is done by replacement of

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the chloride ligand with a linker residue since the two chloride ligands are displaced with

water via ligand exchange reaction when placed in an aqueous medium. An example is the
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conjugation of platinum(IV) agents to a SWNT by carboxylate chelation method.[56] The

uptake by the cancer cells takes place due to endocytosis process where pH is less than seven
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inside the endosomes and lysosomes. This drop in pH enhances the reductive release of the

platinum (II) core complex. Due to the high cytotoxicity of SWNT-Pt to cancer cells, the
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activity increases by 100-folds compared to platinum free drug. This performance is due to
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the more efficient endocytic uptake and enhanced activation by the drug attached to the
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SWNT nanocarrier. Different from platinum-based chemotherapy drugs, SN38 is an active

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product in the metabolism of irinotecan which has shown great potential as anti-tumour drug

in targeted drug delivery due to its insolubility in water. PAMAM terminated with carboxylic
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acid group was also used as a carrier for SN38 by attaching with ester linkers and glycine or

β-alanine spacer at the C20-OH position.[57] The small difference in structure by a CH3

group between glycine and β-alanine spacer affects the conjugates activity as well as the rate

of drug release. The release of SN38 with glycine is higher than that with the β-alanine

causing SN38 with glycine to be more potent. Recently, Gu et al. reported the controlled

release of SN38 via thiolysis in the presence of GSH (glutathione) or via enhanced hydrolysis

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due to ROS (reactive oxygen species)-oxidation of the linker, giving rise to high in vitro

cytotoxicity and in vivo anticancer therapeutic activity (Figure 5).[58] By tuning the length of

the OEG chain in the prodrug, the obtained amphiphilic conjugates were capable of self-

assembling into nanocapsules. In vivo studies showed the conjugated SN38 had higher

tolerance than the free drug and gave rise to higher in vitro cytotoxicity and in vivo anticancer

therapeutic activity. Immunosuppressive agents like tacrolimus (FK506) and cyclosporine are

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important therapeutic drug in organ transplantation. FK506 is more potent than cyclopsporine

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by two orders under in vitro conditions, however it is also rapidly cleared in plasma after

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intravenous administration and highly metabolized by P450 3A liver cytochrome enzyme.

Thus to control the pharmacokinetics and enhance the therapeutic efficiency, Hashida et al.
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developed a delivery system using dextran to transport FK506 by conjugating with ester

linker. Owing to the fairly stable ester bond, the rate of drug release became lower in a
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phosphate buffer of pH 7.4 with a half-life of around 150 hours and an extended circulation

time in the bloodstream.[59] Abdi et al. created a targeted delivery system to carry
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cyclosporine by coprecipitating cyclosporine-tethered polylactide with PEG linked

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polylactide.[60] The nanoparticles are internalised in the dendritic cells and move to lymph
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nodes where the cyclosporine is released in a sustained manner when ester linkage is broken
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causing the T-cell proliferation to be suppressed.[60]

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Figure 5. Structure of GSH and the ROS-responsive conjugate of SN38. The conjugate self-

assembled as nanocapsules that can release SN38 with higher in vitro cytotoxicity and
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anticancer therapeutic activity. Reproduced from ref [58] with permission.

2.1.2. Hydrolysis of amide bond

In addition to the hydrolysis of ester bonds, amide linkers are also commonly used as a

conjugation between the drug and nanocarriers due to the ease of functionalising the drug

molecules with an amine or carboxylic acid group and the stability of amide bonds to

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chemical hydrolysis compared to ester bond.[61] However, this stability causes the amide

bonds to be rarely chemically hydrolyzed under physiological conditions, instead harsh

conditions like higher temperatures and the presences of strong acid or base catalysts is

required. This gives it a better pharmacokinetic profile due to the longer circulation duration

in the bloodstream.[62]

Generally, most amide cleavage is done using enzymatic mechanisms by hydrolytic proteases

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like serine proteases, cysteine proteases and zinc-dependent endopeptidases.[63] These

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enzymes are found in various sites like extracellular environment and cell membrane. The

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site of drug release is closely linked to the site of specific action. For instance, the matrix

metalloproteinases (MMPs) such as collagenases are secreted by zinc endopepridases in

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extracellular matrix (ECM) due to degradation of proteins.[64] In some of the tumour cells,

the MMPs are overexpressed and implicated in the dysregulation of angiogenesis causing the
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growth and spreading of tumour cells.[65] By using a MMP-specific peptide as the cleavage

bond, controlled drug release can be obtained at ECM tumour site. In addition, prostate-
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specific antigen, PSA, is from the serine protease family. It is expressed at high levels in
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prostate tumour tissue and also used in controlled drug release as an enzyme target. The
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sequences in the peptide consisting serine (S)-glutamine (Q) and glutamine (Q)-leucine (L)
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are most susceptible to PSA.[66] The peptide spacer comprising SQ dipeptide sequence in

aminoglycoside is conjugated to doxorubicin which is readily released by PSA through

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peptide cleavage.[66] Prostate-specific membrane antigen which is a zinc endopeptidase, is

another enzyme biomarker that is expressed in large amount in prostate cancer cells.[67] On

small molecule substrates like folate-γ-polyglutamate (FA-En), methotrexate-γ-polyglutamate

(MTX-En) and neuropeptide N-acetyl-L-aspartyl-L-glutamate (AC-DE), the PSMA can act as

a glutamate carboxypeptidase (GCPII) by having it in the linkage will allow possibility of

targeted drug release in prostate tumours. Another protease that can be part of the peptide

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linkage conjugated to the drug is plasmin.[68] Plasmin similar to PSA is a serine protease

which is found in plasma and lysosomes.

On the contrary, there is no general mechanism for drug release through cleavage of amide

linkage by chemical hydrolysis but some classes of specialised linkages conjugated to the

drug by amide linkage are responsive to low pH environments.[69] The specialised linkage

contains a form of maleic acid like citraconyl, cis-aconityl and maleyl group, it is cleaved in

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an acid-catalyzed mechanism to release the drugs. At pH 7, this linkage is very stable and

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does not hydrolysed much even after four days of incubation. However, the rate of hydrolysis

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will increase with increasing acidity, this allows controlled realise of the drug by varying the

pH of the environment. Previous study has shown that the cis-aconityl linker hydrolysed
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significantly faster than the maleyl linker in the same environment while citraconyl linker are

slightly faster than cis-aconityl linker.[70]

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Several anticancer drugs such as MTX, DOX and α-Tocopheryl succinate have been

conjugated onto different delivery carriers and controlled drug release profiles were achieved
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by the hydrolysis of the amide bond. For example, methotrexate (MTX) has been used in
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several cancer targeted treatment through conjugating with a peptide based amide linkage.
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The drug conjugation is done by having an amide spacer that is peptidase susceptible. The
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drug release is controlled by the mechanism that depends on the overexpressed peptidases on

the cancer cell surface. Recently, a dextran-based delivery system is designed by Langer et al.
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to deliver MTX that is peptide linked and susceptible to matrix metalloproteinases

(MMPs).[71] The efficiency of drug release is sensitive to the type of peptide space as well as

MMP subtype. A lower level of release (61%) in 24 hours is observed for treatment done

using MMP 9 and MTX is not released when it is directly conjugated to dextran or by

random peptide sequence.[71] Similar to dextran, the MMP specific peptide spacer is used in

FAR-targeted MTX delivery through PEG-poly(lysine)-grafted dendrimers. The peptide

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linked MTX is also used with MWNT and hyaluronic acid (HA). Bianco et al. used MWNT

that was attached to MTX via amide-based spacer with varied peptide length and sequence

and the uptake by the breast cancer cells was based on folate receptor-mediated endocytosis.

Results showed that only specific conjugates that are linked by GFLG peptide sequence could

inhibit cell proliferation.[72] In another example, Homma et al. used a small set of short

peptides for conjugation between MTX and HA backbone for the treatment of osteoarthritis

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as MTX is a general cytotoxic chemical for cancer and rheumatoid arthritis treatment. The

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results also showed the need for specific peptide for MTX release.[73]

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Doxorubicin is conjugated by amide or peptide based linkers that are made by attaching

daunosamine sugar residue so that it is cleaved by peptidase action or low pH. Jones et al.
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used peptide containing LSQ sequence that is hydrolysed between S and Q site by prostate-

specific antigen (PSA) to release doxorubicin.[74] When expose to prostate tumour cells that
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secrete PSA, the drug release will produce free doxorubicin and doxorubicin-leucine. This

allows tumour selective drug delivery as the doxorubicin is more cytotoxic to cancer cells
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secreting PSA than those that do not.

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In a recent study, Zhang et al. demonstrated the fabrication of DOX-Fe3O4 conjugates with
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the ability to release the conjugated drug by cleavage of amide bond in weakly acidic
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environment (Figure 6).[75] The DOX-Fe3O4 nanoparticles were further embedded in a

polyethylene glycol (PEG) functionalized porous silica shell (Fe3O4-DOX/pSiO2-PEG) to

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achieve the controlled drug release. In addition to the cleavage of chemical bond, the

presence of a porous silica shell could provide a protective layer for drug molecules and

magnetite nanoparticles. Furthermore, the porous silica shell imposed a further obstacle for

the DOX release from the carrier, and thus it exhibited a slower release rate than the uncoated

DOX-conjugated Fe3O4 nanoparticles. The bioassay showed that when the nanoparticles were

transported to early endosomes, the endosomes could fuse with low pH lysosomes to cleave

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the amide bond between DOX and the particle. This allows the DOX to be released first from

the Fe3O4 particle surface and then to enter via the pore channels into the nucleus of the target

cell, where it performs antitumor activity.[75]

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Figure 6. Schematic representation of the synthesis of the magnetic drug delivery system
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composed of Fe3O4-DOX conjugation cores and a PEG-functionalized porous silica shell.

Reproduced from ref [75] with permission.

α-Tocopheryl succinate, α-TOS, is a vitamin E prototype that has a potent apoptotic activity

and showed possible of being an adjuvant for cancer immunotherapy.[76] It consists of a

chromanol substituted with a long hydrophobic tail which causes it have poor solubility in

water, poorer biocompatibility and thus limited use in cancer chemotherapy. Recently, Shi let

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al. used FA-conjugated G5 PAMAM dendrimers conjugated to α-TOS with five to ten α-TOS

molecules with one dendrimer through succinyl amide bond.[77] This new medicine is water

soluble and uptaken by FAR(+) cancer cells causing apoptosis in both vitro and vivo test. The

release mechanism of the drug is possibly due to the cleavage of one or two linkage

connecting the ester linker to α-tocopheryl or amide linker to dendrimer. Other amide linkers

in peptides like oligo(gycine) for camptothecin (SN38) and valine-citruline peptide for

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auristatin were also used for control drug delivery since they are cleavable by less specific

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proteases like cathepsin B which are found abundant in lysosomes. This allows the possibility

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of cell specific drug delivery for cancer treatment.
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2.1.3. Hydrolysis of hydrazone bond

Hydrazone based linkers are the linkers that are terminated with acyl hydrazone, alkoxyl
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carbonyl hydrazone and benzenesulfonyl hydrazone. Many different classes of anticancer

therapeutic molecules like doxorubicin, paclitaxel, auristatin, calicheamicin and platinum-

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based agents make use of hydrazone linkage for conjugation.[20] However, these drug
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molecules or derivatives usually contain chemical handle like ketone or aldehyde moiety that
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can conjugate with the hydrazine terminated linker. For example, Wang and his co-workers
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recently reported a dual pH-sensitive polymer–doxorubicin nanoparticle system for efficient

anticancer drug delivery (Figure 7).[78] In this study, the as-developed nanoparticulate
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system could increase the intracellular drug concentration which is a major challenge for

cancer therapy. At tumor extracellular pH (∼6.8), the nanoparticle is capable of reversing its

surface charge from negative to positive to facilitate cell internalization. Subsequently, with

the significantly increased acidity in subcellular compartments such as the endosome (∼5.0),

the doxorubicin release from the endocytosed drug carriers was achieved by the cleaving the

hydrazone bond. The cell culture results showed that the combination of the tumor

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extracellular and endo-/lysosomal pH responsivenesses could provide a versatile approach for

efficient cancer chemotherapy.[78]

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Figure 7. Chemical structure of the dual pH-responsive polymer–doxorubicin (DOX)
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conjugate (PPC-Hyd-DOX-DA) and schematic illustration of its pH triggered cellular

internalization and intracellular drug release through the cleaving the hydrazone bond.
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Reproduced from ref [78] with permission.

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It has been noted in previously study that the hydrazone linkages are not reactive to
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hydrolysis at physiological pH condition and during systemic circulation but becomes

susceptible at lower pH.[20] Since the pH in extracellular tumour microenvironment is more

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than the optimal pH of less than 5, the drug release is slower compared to the acidic

environments in endosomes and lysosomes as well as during endocytosis in the tumour cell.

As for the synthesis, there are general two approaches have been well established for

hydrazone conjugation of drug molecules with a carbonyl group as shown in Figure 8.[79]

The first method involves nanocarrier with hydrazine-terminated linker integrated in before

the conjugation to the drug molecule.[80] This method is usually used for drug molecules

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with nucleophilic groups like amine in the structure. However, if the nanocarrier contains a

non-hydrazine functional group like carboxylic acid then the amine from the drug may have a

high tendency to react and conjugate with the nanocarrier to form amide linkage instead. This

chemically stable amide linkage formed will cause the drug to remain conjugated even in

acidic condition. The second method involves the hydrazone linker to be integrated in the

drug molecule through the reaction of hydrazine linker before reacting with the carrier by

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chemo-selective reaction.[81] This method was used in paclitaxel conjugated to a

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bifunctional linker with a maleimido and acyl hydrazine group.[82].

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Figure 8. Synthetic methods for conjugation of drug using hydrazone linkage. Reproduced

from ref [79] with permission.

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2.2. Cleavage by disulfide exchange

Disulfide linkers are one of the primary linkers used in the conjugation of targeted drug.[83]

It does not undergo cleavage by hydrolysis but cleavage through disulfide exchange reaction

or by obtaining thiol in an electrochemical reduction reaction.[82] The chemical mechanism

of clavering is activated by endogenous thiol molecules like cysteine, N-acetylcysteine,

glutathione (L-γ-glutamyl-L-cysteinyl-L-glycine, GSH), homocysteine, thiolglycolic acid as

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well as other peptides containing cysteine.[84] Most of the disulfide cleavage occurs in the

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cytoplasm after endocytosis, this allows the linkers to be sufficient stable for the nanocarriers

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linked to the drug to undergo targeted intracellular uptake. The drug release is affected by the

thiol activated intracellular mechanism and not the redox reaction on the cell surface.
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Amongst the endogenous thiol molecules, GSH plays an important part in drug release in

specific cancer cell. GSH exist mainly in reduced form within the cell like the cytoplasm,
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mitochondria and nucleus.[85] GSH is involved in cellular detoxification mechanisms where

GSH is conjugated to cytotoxic xenobiotics or chemotherapeutic agents or in redox reactions

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with genotoxic reactive oxygen species, this causes a high GSH concentration in cytoplasm.
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The cellular expression level can be adjusted to as high as 20 to 14 mM.[20] The high
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reactivity and expression of GSH allows it to be used as a mechanism for controlled drug
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release at targeted cancer cells. In addition, GSH is often used to reduce the xytotoxicity of

anticancer therapeutics in malignant cells but it affects the resistance of these cells towards
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some type of drugs. There are some anticancer therapeutic agents in the market using

disulfide linkage for drug delivery. Instead of direct linkages between the drug molecules and

nanocarriers, the disulfide attachment of anticancer drug molecules with nanocarriers or

antibodies that are cancer specific are done indirectly as the drug molecules do not have free

thiol or disulfide groups in their chemical structure. The mechanism of controlled drug

release is done by proper integration of the linker in the spacer. This mechanism is used on

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anti-tumour and anticancer chemotherapeutic agents like taxoid, paclitaxel, cisplatin,

gemcitabin, doxorubicin, maytansine, mitomycin C and calicheamicin.[67] For example, by

using paclitaxel-disulfide linked molecules (PTX-SS-1, PTX-SS-2, PTX-SS-3), the disulfide

spacer is attached to paclitaxel by carbonate or ester linkages at C7, C10 or C23 position of

the side chain to enable the mechanism of free drug release as GSH will activate the release

of the drug molecule terminated with a thiol moiety in a disulfide exchange reaction.[74] The

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ester or carbonate group are attacked by the nucleophilic thiol causing the drug intermediate

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to undergo thiol-mediated intramolecular cyclisation reaction forming 2-oxathiolone (PTX-

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SS-1) or five membered thiolactone (PTX-SS-2) while releasing paclitaxel. Ojima et al. used

the same mechanism for paclitaxel on taxoid by conjugating it with an anti-EGFR mAb.[67]
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These molecules are 2 to 3 orders of magnitude more effective than docetaxel or paclitaxel

and displayed high anti-tumour efficacy on paclitaxel resistant tumour in in vivo studies.[67]
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In a recent study, Huo et al. designed a nanosized micelle system self-assembled from

polymer–drug conjugates which exhibit conspicuous physical drug-loading and sufficient

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drug release in targeted areas (Figure 9).[86] The desirable disulfide exchange of the micelles
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changes along with the micelle morphology and drug release rate at different reducing
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conditions. In addition, enhanced intracellular uptake of the polymer–drug conjugates and

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accelerated drug release in the cytoplasm were observed, thereby leading to an enhanced

cytotoxicity and higher apoptosis rate.[86]

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Figure 9. Illustration of the self-assembly, accumulation at tumor tissue and intracellular

trafficking pathway of redox-sensitive HA–ss–PTX micelles. The intracellular trafficking

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pathway includes steps of receptor-meditated cellular internalization, endo/lysosomal escape,

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reduction triggered micelle disassembly, and drug release. Reproduced from ref [86] with
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permission.
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2.3. Cleavage by hypoxia activation

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Hypoxia is the state where there is abnormally low oxygen supply in the cells and tissues.

Due to the poor perfusion cause by growth of new immature vessels results in deprived of

oxygen and leads to formation of solid tumours (Figure 10). The tumour microenvironment

usually has low oxygen levels which causes the shift of the equilibrium of the enymatic

activities of oxidoreductases towards substrate reduction favoured activities. Based on this

theory, selective targeting of tumour cells by the use of prodrug and substrates that are

activated only by oxidoreductase or enzymes that are active in hopoxic environment.[87]

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This enzyme activated mechanism was used with anticancer therapeutic agents like

apaziquone (E09), mitomycin C, banoxantrone (AQ4N), TH-302, RH1 and PR104A.[20] The

hypoxia linkers are cleaved during hypoxia catalyzed by oxidoreductases and the prodrugs

undergo reductive activation resulting in the release of drugs in the tumour. These linkers are

classified into different groups like quinone-trimethyl lock systems, nitroaromatic

heterocyclics, indolequinone and N-oxides based on the core functionality of the

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substrate.[20] The symbolic hypoxia linker, quinone-trimethyl lock systems releases the drug

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via two electron reduction mechanism when incubated with chemical agents like NaBH4 and

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Na2S2O4.[88]
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Figure 10. Schematic presentation showing the hypoxic tumour microenvironment and a
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generalised scheme for the mechanism of hypoxia-activated prodrugs by one- and two-

electron reductases under aerobic and hypoxic conditions. Reproduced from ref [89] with

permission.

Hydroquinone linkers are cleavage by stronger reducing agents like cytochrome P450

oxidoreductase (POR) through two electron reduction mechanism. The intermediate formed

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is subjected to intramolecular six-membered-ring cyclization causing the drug molecules to

be released.[90] Ideally the rate of drug release is affected by the drug linker's conformity, if

the position is favourable for intramolecular attack, rate of drug release will increase. The

efficiency of drug release activated by hypoxia is determined by the chemistry and the design

of the linker system as well as the physiological factors. For example, inside the tumour, the

condition is hypoxic and poor blood perfusion takes place causing a reduction of the drug

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exposure level to the tumour cells. On top of that, hypoxia can take place in ordinary cells

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which can causes specific drug release to tumour cells to decrease. In order to overcome this

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issue, nanocarriers which are tumour cell specific can be attached to hypoxia activated linkers

to increase specific drug release.

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2.4. Other cleavage approaches in control drug delivery
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There are some other cleavage approaches have been explored in control drug delivery. For

example, mannich base are the product of condensation reaction aided by aldehydes between
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nucleophilic molecules like amines, phenols, carboxamides and ketone with primary or
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secondary amine molecules.[91] It is used as a prodrug that improves pharmacokinetics and

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solubility of the drug molecule. For example, Doxorubicin can be conjugated using linkage
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from formaldehyde to ligand, Arg-Gly-Asp (RGD) peptide that targets the αvβ3 integin

receptor for tumour targeting.[92] Synthetically, Mannich base systems have advantage of
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being chemo-selective to amine, not forming linkers in the presence of various functional

groups and also the aqueous-alcoholic reaction condition which allows dissolving of charged

and polar drug molecules.[93] In doxorubicin drug delivery for prostate cancer cells, the drug

release mechanism is done by cleaving Manich linker through hydrolysis of imine Schiff base

in the cytosol. This doxorubicin is more cytotoxic in drug resistant and drug sensitive tumour

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cells compared to unmodified one due to the covalent modification of DNA base pairs

through doxorubicin Schiff base.[94]

In addition, self-immolation reaction is the sudden and rapid spontaneous intramolecular

reaction that is activated by an applied external stimulus.[95] The self-immolation linkers

have a dual function in the structure since the triggering moiety is linked to a self-immolative

spacer instead of the directly to the drug, this causes the drug release mechanism to vary from

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the usual direct linked drug (Figure 11).[95, 96] It shows that self-immolation reaction could

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lead to the scission of bonds at the proximal end of the spacer, with an end result being the

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release of the conjugated cargo. Each of the triggering moiety are cleavage by certain

external stimulus like enzymes, thiol-disulfide exchange, low pH, bioreduction and light and
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this activates the free spacers and electronic cascade reaction or spontaneous cyclization will

take place releasing the drug.[97] These electronic cascade reactions are based on carbamate
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fragment, elimination reaction, cyclization of amine-terminated spacer to a five membered

urea or cyclization of trimethyl lock spacer to lactone moiety.[98] There are several
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advantages of using self-immolative release mechanisms in targeted drug delivery.[99]

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Firstly, there is intensification of the frequency of release through continual drug release for
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every cascade reaction. Secondly, efficient cleavage of the trigger groups by macromolecular
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enzymes can take place since there is a relief from steric congestion between drugs and

trigger moiety due to longer spacer length. Lastly, the introduction of self-immolative linkers
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allows potential unstable drugs to be conjugated to the nanocarriers.[100]

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Figure 11. Schematic illustration of the concept of self-immolative linkers (SIL). Removal of

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the protective group at the distal end of the self-immolative linkers is followed by a fast

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release of the drug at the proximal end of the spacer. Reproduced from ref [95] with

permission.
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Photochemistry is applied in drug release by applying light to a photocage containing a

temporarily inactivated drug molecule. Photocage is made of a photon-cleavable trigger

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group which is cleavable by light allowing the drug molecules to be released in a controlled
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manner.[101] This method can be used for drug molecules like pacitaxel, doxorubicin,
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methotrexate, camptothecin etc. The common linkers are mostly UV light responding
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aromatic rings like o-nitrobenzyl (ONB), coumarin, xanthene, benzophenone and

quinoline.[102] For example the coumarin linkers consist of drug molecule that is covalently
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bonded to a methyl group at the C-4 position of coumarin, they are cleaved by one photon or

two photon excitation mechanisms. This two photon mechanism has a greater cross section

for uncaging causing a more efficient drug release compared to the ONB one.[103] The rate

of drug release trough photo-reliable linkers is affected by the wavelength of light, excitation

mechanism, exposure time and pH condition. At acidic or basic condition, the rate of release

is faster than that of neutral pH due to the change in molar absorptivity of the linkers or/and

the leaving group effect of the drug.[104] Light controlled drug release is used in anticancer

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therapeutics like fifth generation (G5) PAMAM dendrimer which are conjugated to a folate

ligand that works as a targeted carrier for the doxorubicin that is photocaged.[105] The folic

receptor (FAR)-overexpressing KB cancer cells can take up the non-cytotoxic conjugated

drug through FAR mediated endocytosis and the doxorubicin is released causing inhibition of

cell proliferation under exposure to UV light. This method allows the doxorubicin to undergo

targeted cytotoxicity to cancer cells.[105]

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Recently, some chemical linkers like Au-S bond and diazo bond with a spacer group joined to

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the drug molecule go through thermal cleavage releasing the drug as they are unstable under

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thermal stimulus.[106] The main concerns with this mechanism are the difficulty of precise

application of heat to specific cells and the severity of damage caused by temperature higher
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than physiological range. To avoid these issues, specialised nanoparticles that releases heat

upon application of irradiation can be used. These nanoparticles conjugated to the drug can
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be taken up by the specific cells, heat is released near the drug conjugated nanoparticles when

irradiation is applied causing controlled drug release. Reidinger et al. used this mechanism on
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iron oxide nanoparticles(IONPs) which is conjugated to doxorubicin by azo linker on KB

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cancer cells (Figure 12).[106] The IONPs generates heat when exposed to alternating
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magnetic field (AMF) causing drug release and cytotoxicity on KB cancer cells. The azo
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linkers can be cleaved through mechanism activated by biologically or thermolysis. The

biological mechanism is used to activate anti-inflammatory azo-linked prodrugs of 5-

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aminosalicylic acid (mesalazine; ASA) like balsalazide, sulfasalazine, olsalazine or

ipsalazide.[107] Sulfasalazine is used in therapeutic drug in colon since the azo linker

undergoes reductive cleavage by bacterial enzymes in the colon producing an active

product.[108] It is noted that the immobilization of azo-group is possible to load and release

both hydrophilic and hydrophobic drugs. This technique also allows reduction of mass ratio

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between the drug molecules and nanovehicles. Selective release can also be achieved by

binding the drug molecules at various specific distances from the nanoparticles.[19]

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Figure 12. Thermal cleavage of azo linker to release doxorubicin. Reproduced from ref [106]

with permission.
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3. Drug release mechanisms through nanocarrier control

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Instead of cleavage of covalent conjugation, drug loading and drug release can also occur
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through non-covalent mechanisms. Examples of such mechanisms are complexation

interactions with nanocarriers or physical encapsulation. The biological activity of the

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nanocarriers and the rate of drug releases is greatly affected by the surface chemistry,

physicochemical properties and physical properties like particle size and shape of the
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nanocarriers as well as the density of the drug binding sites since these affect the adhesion of

cell surface, phagocytosis and degradation process. Surface modification of the nanocarriers

can also improve biocompatibility and enable drug complexation as well as host-guest

interactions. Common nanocarriers used for drug release through carrier control are

dendrimers, polymers and liposomes etc. These nanocarriers undergo the encapsulation

mechanism by altering the pore size of encapsulate, the effective volume, crosslinks,

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confirmation and formation of microbubbles through physicochemical stimuli like light,

change in pH or ion gradient, reaction with thiols, higher temperature and ultrasound

exposure etc.

3.1. Typical nanocarriers for drug encapsulation

As discussed above, there are many encapsulation methods that are used for drug delivery,

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some of the typical types like layer-by-layer self-assembled capsule, micelles, liposomes,

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nanogels and polymersomes will be covered as the nanocarriers in the next section. To enable

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more effective targeted drug delivery, these capsules can be coated with specific antigen. One

of promising encapsulation method involves electrostatic layer-by-layer (LbL) self-assembled

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procedure where the opposite-charged components are adsorbed alternately on the surface to

form a nanoshells. The charged components used can be linear polyelectrolytes, antibodies,
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enzymes or inorganic nanoparticles.[109] The core needs to be insoluble in certain conditions

like low pH and soluble in conditions where controlled release is needed. In general, the
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release rate is affected by the type of coating and the thickness of the encapsulation. An
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alternative encapsulation approach is drug loading into micelles. Micelles are a widely
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discussed nanocarrier that can encapsulate the drug. It is made up of amphiphilic

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macromolecules that have two distinctive block domains that are hydrophobic and

hydrophilic as well as copolymers that are in di-blocks, tri-blocks or grafted. When these
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micelles are exposed to water, the hydrophilic and hydrophobic blocks will undergo phase

separation, forming a nanoscopic supramolecular core-shell structure.[110] The micelles can

have different structures like sphere, rod, vesicle, tubule or large compound micelles which

will affect the pharmacokinetic properties of the nanocarrier (Figure 13).[111] Sutton et al.

uses the polymer micelles loaded with doxorubicin and paclitaxel for targeted drug release on

tumour cells.[110]

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PT
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Figure 13. Schematic illustration of polymer micelle with different morphologies: (a)

spherical, (b) wormlike rod, (c) vesicle, and (d) large compound micelles. Reproduced from

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ref [111] with permission. NU
Liposome is a vesicle consisting of at least one lipid bilayer and can be used as a nanocarriers
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for therapeutic drugs.[112] They are usually made up of phospholipids. They make use of

contract facilitated drug delivery which involves the lipid monolayer of the carrier binding or
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interaction with specific cell membrane that will enhance the lipid-lipid exchange and
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increase diffusion of drug into the cells. Similarly, polymersomes are hollow shell
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nanoparticles made up of two layers of synthetic polymers with many same chemical features.
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The drug loading and release make use of the block copolymers with thick membrane

together with their aqueous lumen as well as the pH sensitivity. This nanocarrier allows
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doxorubicin and taxol to be efficiently transported to the targeted tumour cells, in fact when

these drugs are encapsulated in polymersome, they will spontaneously self-assembled to form

a cocktail that decreases the proliferation of tumour cells than without the polymersome.[113]

In another aspect, nanogels which refers to hydrogel nanoparticles are used for encapsulation

of drugs in therapeutic drug delivery. They are usually made up of hydrophobic

polysaccharides possess the properties of hydrogel like hydrophilicity, flexibility, high water

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absorptivity, versatility and biocompatibility.[114] Therefore, nanogels are useful for drugs

that have poor solubility. The control release system of hydrogel are usually time controlled

or stimuli responsive. The stimuli can be physical like temperature, electricity, pressure,

sound, light, or magnetic field, it can also be chemical like pH, ions, solvent composition or

specific molecule activated.[10, 115]

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3.2. Factors for control drug release from nanocarriers

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The controlling of the size of pores in the nanocarriers is one of the improtant mechanisms to

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control the drug molecules that is released. Gao et al. used hollow mesoporous silica

nanoparticles (HMSNPs) of pore sizes, 3.2, 6.4 and 12.6 nm to show how the rate of drug
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release and diffusion of doxorubicin is related to the pore size.[116] The pore size is used to

alter the duration of drug release. Another essential factor of nanocarriers in control of drug
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release is the effective volume. The change in effective volume of the nanocarrier through

use of external stimuli can control the opening or closing mechanism of the vehicle allowing
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control drug delivery to take place. This can be applied to drug encapsulating nanosystem
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made of liposomes and inorganic nanoparticles through the lowing the loading capacity after
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cancer cells uptake by decreasing pH, temperature changes or ultrasound exposure. In the
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liposome system, the local density of the polymer left on the liposome surface increases with

protonation of the polymer side groups, this could cause liposomal membrane to collapse and
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release the drug. For example, the anticancer agent, arsenic trioxide (As2O6) loaded into the

poly(acrylic acid)-grafted liposomes uses the mechanism mentioned above.[117] In addition,

the reduction of crosslinks that is used for drug encapsulation and stabilizing the inner part of

nanoparticles controls the change in effective volume and internal permeability of the

nanocarrier.[118] This mechanism is particularly useful in disulfide crosslinked polymer

micelles for controlled release of doxorubicin and methotrexate.[119]

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More importantly, the internal conformation and permeability of a drug delivery system can

also be adjusted by a proper trigger to achieve the controlled drug release.[120] For example,

the photocleavable lipid with ONB incorporated is used to make liposomes that release the

load under light.[120] In addition to the permanent cleavage of the linker, the light also cause

a reversible change in the linker configuration which results in a volume for encapsulation to

decrease and faster drug release rate. Other stimuli like pH can also affect the conformation

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of the nanocarriers. For example in the albumin bound paclitaxel delivery system, it involves

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the albumin receptor mediated intracellular uptake and activation of drug release by the

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denaturing of protein due to low pH in endosome that results in change in conformation.[121]

The non-protein carriers like polymer nanoparticles, sterically stabilized liposomes and
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polymeric micelles which contains pH responsive block copolymers like poly(lactic acid-b-

PEG-folate) or poly(histidine-b-PEG-folate) will also undergo conformation changes due to

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pH changes. Conformity can also be altered by varying temperature and the using of

ultrasound irradiation.
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In another aspect, nanocarriers like nanoemulsion and polymer vesicles uses ultrasound to aid
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the controlled drug release mechanism and the release rate is related to the ultrasound's
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ability to physically stimulate the different structural components in the nanocarrier. An

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example of such system is the nanoemulsion droplet with paclitaxel and perfluorocarbon

(PFC) as a ultrasound responsive gas.[122] The ultrasound irradiation exposure of the

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nanoemulsion produced microbubbles containing the drug and cause an increase in the

effective volume. The ultrasound can also cause a reduction in size in the case of polymer

vesicles that carries doxorubicin which was prepared by self-assembly of amphiphilic block

copolymer (PEO-b-P(DEA-stat-TMA)).[123] Liposomes that are temperature responsive can

be used as nanocarriers for thermally stimulated drug release mechanism. These liposomes

allows different types of molecules including thermal responsive ones to be concurrently

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encapsulated. Chen et al. use liposomes to encapsulate doxorubicin and ammonium

bicarbonate which decomposes and produces carbon dioxide and ammonia gas bubbles at 42

C.[124] The bubbles produced causes defects in the lipid bilayer structure, thus releasing the

doxorubicin molecules.

4. Conclusions

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The use of nanocarriers in therapeutic delivery systems make it possible for proper delivery

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and release of drugs inside the targeted cells. To achieve effective controlled drug delivery,

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specific molecules, ligands or linkers are needed to aid the cell targeting process and the

mechanism for controlled drug release. It is important to design to ensure cytotoxicity is

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localised at the targeted cells and in controlled amount. The mechanisms of drug release

covered in the paper are based on the drug linker type, how it is cleaved chemically and
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biologically or what stimuli that will activate the drug release. Drug delivery using

nanotherapeutic delivery system is promising as it allows the possibility of using therapeutic

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agents that are poor in solubility like paclitaxel, instable in human body, highly toxic like
E
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doxorubicin and cisplatin or have severe side effects. There are many drugs that are still

undergoing research and clinical trials and many more that failed the trials due to poor
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effectiveness as a cure or too severe side effects.

There are many challenges that needs to be overcome to achieve an optimal design for the
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mechanism of controlled drug delivery. Firstly, each of the drug requires a custom made

linkers that makes difficult to match the correct linker to the appropriate drug molecule and

also difficult to control the release mechanism at the targeted site to an ideal state. Too much

or too little drug release will be an issue for it to be used as a therapeutic delivery system.

Furthermore, if the therapeutic system requires many stages to be synthesized, then large

scale production of the medication will be impossible. Secondly, attaching of many different

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molecules and ligands on the nanocarriers can cause steric congestion and lower cell

selectivity leading to many other concerns. Other issues like poor physicochemical properties,

low pharmacokinetics (PK) and poor tissue penetration also need to be addressed.

Acknowledgements

PT
We acknowledge the support from The Fundamental Research Funds for the Central

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Universities (No. 2662015BQ046) and The National Natural Science Foundation of China

(Grant No. 61505063).

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Highlights
 Recent progress of using nanocarrier systems for drug delivery are discussed
 Controlled drug release through different linker cleavages are summarized
 Insights on the drug release mechanism and factors are also provided.

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