polymeric nanomicelles for sustained delivery of anti-cancer drugs
TRANSCRIPT
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Mutation Research 768 (2014) 47–59
Contents lists available at ScienceDirect
Mutation Research/Fundamental and MolecularMechanisms of Mutagenesis
j ourna l h om epage: www.elsev ier .com/ l ocate /molmutComm uni t y ad dress : www.elsev ier .com/ locate /mutres
olymeric nanomicelles for sustained delivery of anti-cancer drugs
oubeena Jeetah, Archana Bhaw-Luximon, Dhanjay Jhurry ∗
NDI Centre of Excellence for Biomedical and Biomaterials Research, University of Mauritius, MSIRI Building, Réduit, Mauritius
r t i c l e i n f o
rticle history:vailable online 24 April 2014
eywords:nti-cancerolymeric micellesustained delivery
a b s t r a c t
In the first section of this paper, the existing and emerging nanotechnology-based cancer therapies –nanoparticles, drug conjugates, nanomicelles – are reviewed. In a second part, we present our originaland unpublished findings on the sustained release of anti-cancer drugs such as paclitaxel, doxorubicin andcamptothecin using block copolymer micelles [PEG-b-poly(dioxanone-co-methyl dioxanone)]. Copoly-mers with variable lengths of hydrophobic and hydrophilic blocks have been synthesized and successfullyloaded with paclitaxel, doxorubicin and camptothecin anti-cancer drugs, with micelles size in the range
oxorubicinaclitaxelamptothecin
130–300 nm. Drug encapsulation efficiencies varied between 15% and 70% depending on drug and copoly-mer composition. The drug binding constants, which give a good insight into drug encapsulation andrelease, were evaluated from UV spectroscopy as we reported previously for anti-TB drugs. Through vari-ation of the methyl dioxanone content of the copolymer, our systems can be tailored for sustained releaseof the different drugs.
© 2014 Elsevier B.V. All rights reserved.
. Introduction
About 13% of all deaths worldwide were cancer-related in 20087.6 million) and projections show that an estimated 13.1 millioneaths are expected in 2030 [1]. The commonest sites of can-er, based on the latest report by the National Cancer Registry inauritius [2], are in males: colon-rectum (14%), prostate (10.5%),
ral cancer (8%) and lung (9.6%). Among females, breast cancer38%) is the most prevalent site of cancer followed by cancer ofhe uterine cervix (10%), colon-rectum (4.8%) and ovaries (5.6%).
Between 2005 and 2008, there were 2286 new male cancerases registered and 3280 new female cancer cases. Breast cancern females accounted for a total of 1239 new cases and childhoodancers for 96 new cases during that period. There were 1950ale cancer deaths and 1900 female cancer deaths recorded during
he period 2005–2008. A list of fifty anti-cancer drugs is availablen Mauritius for treating various types of tumors. These includenthracyclines, taxanes, antimetabolites, hormonal therapy drugsnd platinum analogs.
In the first part of this paper, we present a mini-review ofhe current status of the main existing therapeutics for cancer
reatment and focus on emerging nanotechnology-based ther-pies for delivery of anti-cancer drugs. In a second part, weresent our original and unpublished findings on the sustained∗ Corresponding author. Tel.: +230 4651347.E-mail addresses: [email protected], [email protected] (D. Jhurry).
ttp://dx.doi.org/10.1016/j.mrfmmm.2014.04.009027-5107/© 2014 Elsevier B.V. All rights reserved.
release of anti-cancer drugs chosen from three different classes– taxanes (paclitaxel), anthracyclines (doxorubicin) and alkaloids(camptothecin) – using block copolymer micelles [PEG-b-poly(dioxanone-co-methyl dioxanone)] as nanocarriers.
2. Cancer therapeutics
2.1. Biology of cancer
Most cancer drug development is now biology driven. In a can-cer cell, several genes mutate and the cell becomes defective. Thereare two general types of gene mutations. One type, dominant muta-tion, is caused by an abnormality in one gene in a pair. An exampleis a mutated gene that produces a defective protein that causesthe growth-factor receptor on a cell’s surface to be constantly “on”when, in fact, no growth factor is present. The result is that thecell receives a constant message to divide. This dominant “gain offunction gene” is often called an oncogene (onco = cancer) [3].
The second general type of mutation, recessive mutation, ischaracterized by both genes in the pair being damaged. For exam-ple, a normal gene called p53 produces a protein that turns “off” thecell cycle and thus helps to control cell growth. The primary func-tion of the p53 gene is to repair or destroy defective cells, therebycontrolling potential cancerous cells. This type of gene is called an
anti-oncogene or tumor suppressor gene. If only one p53 gene inthe pair is mutated, the other gene will still be able to control thecell cycle. However, if both genes are mutated, the “off” switch islost, and the cell division is no longer under control.
48 R. Jeetah et al. / Mutation Research 768 (2014) 47–59
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O
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H2N
HO
Epiru bici n
O
O
O
O
O
O
OH
OH
OH
H2N
OH
Daunorubicin
O
OH
OH
OH
OH
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NH2
Doxorubicin
cheme 1. Normal cell cycle depicting the different stages at which anti-cancerrugs can act, namely the G1 phase, S phase, G2 phase and M phase.
Many forms of chemotherapy are targeted at the process ofell division. The rationale being that cancer cells are more likelyo replicate than normal cells. An understanding of the princi-les of tumor biology and cellular kinetics is helpful to appreciatehe mechanisms of action of cancer chemotherapy. Advancementsn knowledge about the biology of cancer cells and tumors havellowed the development of drugs which can act at specific stagesf cell life (Scheme 1).
.2. Existing therapies and limitations
Commonly used anti-cancer drugs can proceed via three mech-nisms.
. Damage to the DNA of the affected cancer cells. In this categoryare included drugs such as cisplatin, daunorubicin, doxorubicinand etoposide.
. Inhibit the synthesis of new DNA strands to stop the cell fromreplicating, to prevent tumor growth (also called topoisomeraseinhibitors). In this class, we can find drugs such as methotrexate,fluorouracil, hydroxyurea, and mercaptopurine.
. Stop mitosis or the actual splitting of the original cell into twonew cells to halt the progression of the cancer (also called spindlepoison). Drugs such as vinblastine, vincristine and paclitaxel arefound in this category.
The different classes of anti-cancer drugs are now detailed.
.2.1. AnthracyclinesAnthracyclines are derived from Streptomyces percetus var. cae-
ius bacteria and include drugs like doxorubicin, epirubicin andaunorubicin (Fig. 1). They are chemically similar, with a basicnthracycline structure containing a glycoside bound to an aminougar, daunosamine.
They act by (i) inhibiting DNA and RNA synthesis, thus pre-enting the replication of growing cancer cells (ii) inhibitingopoisomerase II, thus blocking DNA transcription and replicationiii) creating iron-mediated free oxygen radicals that damage DNAnd cells membranes. They are often used alone or in combina-ion with cyclophosphamide as first line chemotherapy for breastancer [4]. Anthracyclines present high cardiotoxicity, limiting
heir use. Oxygen free radical formation from reduced doxorubicinntermediates is thought to be a mechanism associated with car-iotoxicity. Doxorubicin has an initial distribution half-life of 5 min5].Fig. 1. Structure of anthracyclines: doxorubicin, epirubicin and daunorubicin.
2.2.2. TaxanesTaxanes were originally derived from natural sources and
include drugs like paclitaxel and docetaxel (Taxotere) (Fig. 2). Theyare referred to as mitotic inhibitors as they disrupt microtubule
function, thereby inhibiting cell division. They are prescribed forthe clinical treatment of various types of tumors but toxicity andside effects are also serious drawbacks of taxanes. Docetaxel is less
R. Jeetah et al. / Mutation Research 768 (2014) 47–59 49
OH
OO
OHO
O
O
HO
O
OO
O
OH
NHO
Paclit axel
OH
OO
OHHO
O
O
HO
OO
O
OH
NH
O
O
ax el
nes: p
hd
a
2
topurhtt
Docet
Fig. 2. Structure of taxa
ydrophobic than paclitaxel and is approximately twice as potentue to a higher affinity for the microtubule binding site [6].
Taxanes in combination with anthracyclines or non-nthracyclines have led to improved treatment [7].
.2.3. Platinum-based chemotherapyCisplatin (Fig. 3) approved in 1978, has become an impor-
ant component in chemotherapy regimens for the treatment ofvarian, testicular, lung and bladder cancers, as well as lym-homas, myelomas and melanoma. Unfortunately its continuedse is greatly limited by severe dose limiting side effects and drug
esistance. Over the last 30 years, 23 other platinum-based drugsave entered clinical trials with only two (carboplatin and oxalipla-in) of these gaining international marketing approval, and anotherhree (nedaplatin, lobaplatin and heptaplatin) gaining approval inPt
NH3Cl
Cl NH3
Fig. 3. Structure of cisplatin.
aclitaxel and docetaxel.
individual nations. Currently there are four drugs in the variousphases of clinical trial (satraplatin, picoplatin, lipoplatin and pro-Lindac) [8]. Platinum drugs present acute shortcomings such asneurotoxicity, nephrotoxicity and myelotoxicity.
2.2.4. AntimetabolitesAntimetabolites are compounds that bear a structural similar-
ity to naturally occurring substances such as vitamins, nucleosidesor amino acids. Generally, antimetabolites induce cell death duringthe S phase of cell growth when incorporated into RNA and DNAor inhibit enzymes needed for nucleic acid production. Their effi-cacy is usually greater over a prolonged period of time, so they areusually given continuously. There are three main types: folic acidantagonists, purine analogs and pyrimidine analogs.
2.2.4.1. Folic acid antagonists. Methotrexate (Fig. 4) competitivelyinhibits dihydrofolate reductase, which is responsible for the for-mation of tetrahydrofolate from dihydrofolate. This is essential for
the generation of a variety of coenzymes that are involved in thesynthesis of purines, thymidylate, methionine and glycine. A criticalinfluence on cell division also appears to be inhibition of the pro-duction of thymidine monophosphate, which is essential for DNAand RNA synthesis.
50 R. Jeetah et al. / Mutation Research 768 (2014) 47–59
N
N
N
N
NH2
NH2N
N
H
O
O
HO
OHO
Fig. 4. Structure of methotrexate.
NH
N
S
H
N
N
6-mercaptop urin e
N
H
N
S
HN
N
H2N
6-thi oguanine
HN
N
N
N
S
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NO2
rcapto
2tagt
2miii
tla
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in cancer prognosis, growth, and/or metastasis. Table 1 lists thetarget-based compounds under clinical trials and a few othersapproved in the United States [11].
NH 2
Fig. 5. Structure of purine analogs: 6-me
.2.4.2. Purine analogs and related inhibitors. These are analogs ofhe natural purine bases and nucleotides. 6-Mercaptopurine (6MP),zathioprine and thioguanine (Fig. 5) are derivatives of adenine anduanine, respectively. They are able to inhibit nucleotide biosyn-hesis by direct incorporation into DNA.
.2.4.3. Pyrimidine analogs. These drugs resemble pyrimidineolecules and work by inhibiting the synthesis of nucleic acids,
nhibiting enzymes involved in DNA synthesis or by becomingncorporated into DNA, interfering with DNA synthesis and result-ng in cell death.
5-Fluorouracil (Fig. 6) is a pyrimidine analog that interferes withhymidylate synthesis and acts on a wide range of solid tumors. Itsimitations include a short half-life (approx. 5 mn), toxic side effectsnd non-selective action against healthy cells [9].
Gemcitabine (Fig. 7) is used in various carcinomas: non-smallell lung cancer, pancreatic cancer, bladder cancer and breast can-
er. It is being investigated for use in esophageal cancer, and is usedxperimentally in lymphomas and various other tumor types. Gem-itabine represents an advance in pancreatic cancer care. HoweverHN
H
N OO
F
Fig. 6. Structure of fluorouracil.
azathio prine
purine, 6-thioguanine and azathioprine.
an important side effect for more than 10 out of 100 patients is adrop in the number of blood cells made by the bone marrow [10].
2.2.4.4. Target-based agents. Recent developments in molecularbiology and an understanding of the pharmacology of cancer ata molecular level have enabled researchers come up with target-based drugs. These are the agents that are pre-designed to inhibitand/or modify a selected molecular marker deemed important
O
OH F
F
N
HO
N
O
Fig. 7. Structure of gemcitabine.
R. Jeetah et al. / Mutation Research 768 (2014) 47–59 51
Table 1Target-based compounds under clinical trials and marketed.
Compound Product name, manufacturer Mode of action Indication
Imatinibmesylate Gleevec® , Novartis Inhibits a specific tyrosine kinase enzyme, the Bcr–Abl fusiononcoprotein
Gastrointestinal stromal tumorand chronic myeloid leukemia
Gefitinib Iressa® , AstraZeneca & Teva Inhibitor of the epidermal growth factor receptor’s (EGFR, or erbB1)tyrosine kinase domain
Non-small-cell lung cancer
Rituximab Rituxan® , Biogen Idec & Genentech Monoclonal antibodytigen on the CD20+ B-cells, causing their apoptosis
B-cell non-Hodgkin’slymphoma and B-cell leukemia
y that binds the cell surface HER2/neu (erbB2) Therapy oferbB2+ breast cancer
Hapmait86rwa
arm
22cclmifsuismbea
2
pigtr
N
N
O
O
HO O
ON
O
N
Irinote can
N
NH
NH
N
H2N
O
O
NH
NH
O
OH
O
OH
O
Folin ic acid
NH2
NH2
Pt
O
O O
O
Oxalipl atin
TD
It binds the CD20 anTrastuzumab Herceptin® , Genentech Monoclonal antibod
receptor
Epidermal growth factor receptor (HER1/EGFR) belongs to theER family of four distinct receptors (HER1/EGFR, HER2/neu, HER3nd HER4). Therapeutic agents that target the HER1/EGFR signalathway, such as small-molecule tyrosine kinase inhibitors andonoclonal antibodies are now advanced in clinical development
nd two are already licensed for use [18]. HER1/EGFR is deregulatedn many solid tumors, making it an attractive target for anticancerherapy [18]. In non-small cell lung cancer (NSCLC), between 43 and3% of tumors overexpress HER1/EGFR, and EGFRvIII is detected in0% of grade 3/4 gliomas, where it is frequently overexpressed as aesult of gene amplification. Against this background, HER1/EGFRas identified as an attractive target for the development of novel
nticancer agents.HER1/EGFR-specific cetuximab (Erbituxe) is the most advanced
nti-HER1/EGFR MAb in clinical development. Cetuximab wasecently approved in the USA and Europe for patients withetastatic colorectal cancer who are refractory to irinotecan.
.2.5. Drug combination
.2.5.1. Folfirinox. Folfirinox (Fig. 8) is a multi-agent cytotoxichemotherapy regime consisting of a combination of leucovorinalcium (folinic acid), fluorouracil, irinotecan hydrochloride, oxa-iplatin. This combination is used to treat pancreatic cancer that has
etastasized [12]. It is a sequential administration of oxaliplatinmmediately followed by leucovorin over 2 h, and then irinotecan,ollowed by a bolus dose of 5-fluorouracil, and finally, a 46-h infu-ion of 5-fluorouracil all given intravenously every other week forp to 6 months [13]. Patients who received Folfirinox lived approx-
mately 4 months longer than patients treated with the currenttandard of care, gemcitabine (11.1 months compared with 6.8onths) [14]. However, Folfirinox is a potentially highly toxic com-
ination of drugs with serious side effects [15]. The serious sideffects associated with the regimen include neutropenia, neurop-thy, and gastrointestinal problems [16].
.3. Drug conjugates
Drug conjugates consist of antibodies to which a highlyotent toxin is attached via a linker and generally administered
ntravenously (Scheme 2). For example, Adcetris, a drug conju-ate, showed remarkable results in eliminating Hodgkin’s diseaseumors or in causing them to shrink. Survival of patients underecently approved FDA Kadcyla against breast cancer is about 25%
able 2rug conjugates on the market and under clinical development.
Product name Manufacturer Description
Kadcyla® ImmunGen/Genetech/Roche Holding Anti-body drugAdcetris® Seattle Genetics Inc
CMC-544 Wyeth
IMGN 901 ImmunoGen Inc
XyotacTM/Opaxio Cell Therapeutics Inc PGA-paclitaxel
ProLindacTM Access Pharmaceutical Inc HPMA-copolymer-
Fig. 8. Components of folfirinox: folinic acid, oxaliplatin, irinotecan and 5-fluorouracil (Fig. 6).
longer compared to commonly used treatment due to increasedbinding to HER2 protein. Table 2 lists major drug conjugates on themarket or under development.
3. Nanotechnology-based approaches
Drug delivery systems have been developed to improve ther-apeutic index of drugs by improving their administration and
Indication Stage
Breast cancer MarketHodgkin lymphoma, anaplastic large cell lymphoma MarketNon-Hodgkin lymphoma Phase IIISmall-cell lung cancer, ovarian cancer Phase IINon small-cell lung cancer Phase III
Pt MelanomaOvarian
Phase III
52 R. Jeetah et al. / Mutation Research 768 (2014) 47–59
S via a lc o the c
ittois1
r
cgitsde
3
rtntTdrs
Scsa
cheme 2. Mode of action of drug conjugates: an antibody is attached onto a toxin
ancer cells where the toxin is separated from the antibody, thus causing damage t
ncreasing the exposure of diseased tissues to therapeutics. Syn-hetic or natural polymeric materials as drug carriers have knownremendous progress with many systems at commercial stage andthers under clinical trials. These systems have to satisfy the follow-ng requirements in a first instance to qualify as suitable carriers:mall size (<150 nm); relatively large surface area; low CMC (order0−5 to 10−6) and high physical stability.
Scheme 3 shows some of the most common drug delivery car-iers.
Generally, the advantages of these drug carriers over theonventional chemotherapy agents include: selective passive tar-eting to tumor sites; increased efficacy and therapeutic index;mproved delivery of hydrophobic molecules; reduced toxicities ofhe encapsulated agent; low accumulation in vital organs and tis-ues; improved pharmacokinetics (reduced elimination, increasedrug exposure time); increased stability via encapsulation, andnhanced intracellular drug delivery to overcome drug resistance.
.1. Mode of action of nanoparticles
Various cancer cells overexpress growth factors that lead toapid formation of vessels. The latter have leaky boundaries dueo the absence of a smooth muscle layer and allow penetration ofanocarriers. Nanocarriers are internalized into the cell throughhe endosome followed by release of the drug into the cytoplasm.hey accumulate in the tumor tissue that lacks effective lymphatic
rainage via the EPR mediated passive targeting (Fig. 9). Nanocar-iers also accumulate in other tissues with leaky endothelial wallsuch as the liver, spleen and bone marrow.cheme 3. Current drug delivery systems consisting of: (i) amphiphilic blockopolymer micelles (inner hydrophobic core and outer hydrophilic shell); (ii) lipo-omes (lipid bilayer enclosing an aqueous core) and nanoparticles (drug moleculesre dispersed in a polymer matrix).
inker and is intravenously administered. This drug conjugate is directed and entersell.
Active targeting occurs through specific binding of ligandsanchored on the surface of nanocarriers onto tumor cell recep-tors such as antibodies, folate or growth factors and cytokines [17].Active vascular targeting is designed to deliver drugs to block pre-existing blood vessels of tumors to cause tumor cell death fromischemia and extensive hemorrhagic necrosis [18]. Active tumortargeting is the specific delivery of drugs to tumor cells.
4. Nano drug delivery systems
4.1. Liposomal anti-cancer drug delivery
Liposomes are well defined lipid and lipoprotein vesiclesthat offer immense potential for targeting drugs to tumors.FDA-approved liposomal preparations of doxorubicin (Doxil),daunorubicin (DaunoXome), cytarabine (DepoCyt), and ampho-tericin B (Abelcet) have proven to be attractive, less toxicalternatives to the conventional drug formulations [20]. Liposo-mal daunorubicin and amphotericin B have shown less cardiacand renal damage, respectively. PEGylated liposomal doxorubicin,Doxil® was approved in 2005 for ovarian cancer, AIDS-relatedKarposi’s Sarcoma and multiple myeloma. It interferes with celldivision, has a long blood circulation period (t1/2 > 48 h) and size∼100 nm. However, it suffers from a major side-effect which ispalmar–plantar erythrodysesthesia (PPE), also called hand-footsyndrome [21].
Table 3 lists the marketed liposomal anti-cancer products. Mostof the encapsulated drugs act as topoisomerase II poison. Liposomes
for combination therapy are also at different test stages as shownin Table 4.Cisplatin analogs have also been encapsulated using liposo-mal technology L-NDDP (AroplatinTM, AntigenicsInc, Lexington,
Table 3Liposomal and lipidic marketed anti-cancer products.
Trade name Drug Company
DOXIL/Caelyx® Doxorubicin Schering-PloughDaunoxome® Daunorubicin Gilead SciencesMEPACT® MTP TakedaMyocet® Doxorubicin CephalonDepocyt® Cytarabine Sigma-Tau pharmaceuticals
R. Jeetah et al. / Mutation Research 768 (2014) 47–59 53
sing pR ger, Im2
Mcllpestt
4
dtesochea
TL
Fig. 9. Passive and active targeting ueproduced and reprinted with permission from Ref. [19], OC Farokhzad and R Lan009 American Chemical Society.
A, USA) was the first liposomal formulation studied in thelinic for the delivery of a cisplatin analog. LipoplatinTM (Regu-on Inc, Mountain View, CA, USA) is another cisplatin-containingong-circulating liposomal formulation. It is composed of soyhosphatidylcholine, cholesterol, dipalmitoylphosphatidyl glyc-rol, and methoxy-PEG-distearoylphosphatidylethanolamine [5]. Aignificant in vitro cytotoxicity up to 1000-fold higher than that ofhe free drug was observed in human-derived ovarian (IGROV-1)umor cells using liposomal cisplatin.
.2. PEG-based systems
Plasma proteins, known as opsonins, can bind circulating drugelivery devices, including nanocarriers, and remove them fromhe circulation within seconds to minutes through the reticulo-ndothelial system (RES) [22]. Imparting a stealth-shielding on theurface of these drug delivery systems prevents opsonins from rec-gnizing these particles, thereby limiting phagocytosis by the RES
ells and increasing the systemic circulation time from minutes toours or even days. Poly(ethylene glycol) (PEG) modification hasmerged as a common strategy to ensure such stealth shieldingnd long-circulation of therapeutics or delivery devices.able 4iposomes for combination cancer therapy.
Formulation Drugs
CPX-351 5:1 cytarabine:dauCPX-1 1:1 irinotecan andCPX-571 7:1 irinotecan:cispLiposomes co-encapsulating 6-mercaptopurine and
daunorubicin6-mercaptopurine
Liposomes co-encapsulating quercetin and vincristine 1:2 quercetin:vincCationic liposomes co-encapsulating siRNA and mercaptopurine
and doxorubicinDoxorubicin, MRPsiRNA
Transferrin-conjugated liposomes co-encapsulating doxorubicinand verapamil
Doxorubicin and v
olymeric micelles and nanoparticles.pact of nanotechnology on drug delivery, ACS Nano, 3(1), 16–20, 2009. Copyright
The protective (stealth) action of PEG is mainly due to the for-mation of a dense, hydrophilic cloud of long flexible chains on thesurface of the colloidal particle that reduces the hydrophobic inter-actions with the RES. The tethered and/or chemically anchoredPEG chains can undergo spatial conformations, thus preventing theopsonization of particles by the macrophages of the RES, whichleads to preferential accumulation in the liver and spleen. PEGy-lation therefore improves the pharmacokinetics of drugs. Table 5lists some of the PEG-based anti-cancer therapeutics on the marketor under clinical development.
4.3. Polymer-drug conjugates
Drug conjugation to a macromolecular carrier has the principaleffect of limiting cellular uptake via pinocytosis [25]. Here, cel-lular internalization of the macromolecule occurs via membraneinvagination, with immediate transfer to the endosomal compart-ment of the cell. A series of vesicle fusion events follow, with themacromolecule eventually being transferred to the lysosomal com-
partment and subsequently exposed to lysosomal enzymes. Thisrestriction of drug uptake to the lysosomotropic route allows theexploitation of the opportunities for both passive and active target-ing of tumors.Indication Status
norubicin Acute myeloid leukemia Phase II floxuridine Colorectal cancer Phase IIlatin Small-cell lung cancer In vivo
and daunorubicin Acute lymphocytic leukemia In vitro
ristine Breast cancer In vitro1-targeted siRNA and BCL-2 targeted Lung cancer In vitro
erapamil Leukemia In vitro
54 R. Jeetah et al. / Mutation Research 768 (2014) 47–59
Table 5PEG-based anti-cancer polymer therapeutics on the market or in clinical development [23,24].
Product name Description Clinical use Route Stage
Polymer protein conjugateNeulasta TM PEG-Human-GCSF Chemotherapy-induced neutopenia SC Market
PEG-drug conjugateNKTR-102 PEG-irinotecan Cancer-metastatic breast IV Phase IIPEG-SN 38 Multiarm PEG-camptothecan derivative Cancer-various
G-lipoposi’s
d[3haitstpelieodBc
4
ap
PEDoxil PEG-liposome doxorubicin KaLipodox
N-(2-hydroxypropyl) methacrylamide (HPMA) copolymer-oxorubicin conjugate (PK1) has achieved success in clinical trials26]. It is hydrophilic and nontoxic in the rat, even at doses of0 g/kg. HPMA copolymer doxorubicin (PK1, FCE28068) (Fig. 10),as a peptidyl linker (Gly-Phe-Leu-Gly) that is stable in the plasmand has been shown to concentrate within solid tumor models. Its then cleaved intracellularly by lysosomal cysteine proteinases,hereby allowing intratumoral drug release. Preclinical work hashown that PK1 demonstrates radically different pharmacokine-ics compared to free doxorubicin, with an increased distributionlasma half-life from 5 min to 1 h. The stable peptidyl linker alsonsures that little or no free doxorubicin is liberated into the circu-ation following i.v. administration, thus increasing the therapeuticndex of the conjugate. In vivo antitumor activity of PK1 has beenxamined using a large panel of model tumors i.p. administrationf PK1 has been shown to display a higher activity profile than freeoxorubicin against the 20pprox.tumor model L1210, melanoma16F10, Walker sarcoma, P388 leukemia, M5076, and the humanolon xenograft LS174 T.
.4. Polymeric micelles
Self-assembled from biodegradable and biocompatiblemphiphilic block polymers and with sizes ranging 10–200 nm,olymeric micelles are promising nanocarriers for anticancer
H2C C
CH3
OC
NH
CH2
CHOH
CH3
H2C
x
C
CH3
OC
NH
CH2
CO
NH
CHCH2
OC
NH
CHCH2CH
CH3
CH3OC
NH
CH2
CO
HN
OHO
H3C
O
HO
CHOH2C
OH
OH
O
O
O
y
O
Fig. 10. Structure of PK1.
somessarcoma, ovarian/breast cancer IV Market
drugs, as attested by a number of such formulations currentlyin clinical trials [27]. Several PEG-based polymeric micelles havebeen approved for clinical trials for chemotherapeutic drugs [28].
The cytotoxicity of drug-free and drug-loaded PEGylatednanomicelles toward a number of cell lines has been determinedin vitro by several researchers (Table 6). Some interesting resultsare here discussed. Diao et al. [29] found that doxorubicin-loadedPEG-PCL had greater cytotoxicity than pure doxorubicin solutiontoward 21pprox.21in-resistant K562 and even had the ability toreverse multidrug resistance. A similar result was obtained by Yooet al. [30] with PEG-PLGA micelles encapsulating doxorubicin. Sincecell types vary in their response to nanoparticles, it is usually rec-ommended that cytotoxicity assays should not be restricted tosingle cell study [31]. For example, paclitaxel-encapsulated PEG-PCL-PEG nanomicelles tested by Zhang et al. [32] elicited differentresponses toward different cells. HepG2 cells showed less sensitiv-ity than EMT6 cells as the HepG2 liver cells are able to metabolizeand detoxify paclitaxel. Cytotoxic effect also varied with pH fordoxorubicin-loaded PLA-PEG-polyHis nanomicelles; the cell via-bility was found to decrease with decreasing pH: pH 7.4–87%, pH6.8–40%, pH 6.4–30%, pH 6–26% [33]. Similar results were obtainedin the case of camptothecin-loaded MPEG-PAE, which showedmore cytotoxic effect at pH 6.4 than at pH 7 [34].
Thambi et al. [35] synthesized a novel PEG-PBLG copoly-mer by introducing a disulphide bond. The cytotoxicity ofcamptothecin-loaded PEG-SS-PBLG micelles to SCC7 cells wasmuch greater than that of camptothecin-loaded PEG-PBLG micelles.This was attributed to the rapid release of camptothecin fromthe bioreducible micelles by facile cleavage of disulphide bond.Paclitaxel-loaded PEG-P(CL-co-TMC) micelles inhibited better cellviability than Taxol at a concentration of 12.5 �g/ml of PTX. Cre-mophor EL, used to solubilize paclitaxel in the Taxol formulation,reduced cell viability to 59% and 54% when its concentration was 2.2and 1.1 mg/ml, respectively after 24 h of incubation. On the otherhand, the drug-free micelle killed only 18% of cells [36].
With promising results obtained on cell lines, some systemshave moved to the next stage of clinical trials. Table 7 shows someof the polymeric micelles at different stage of clinical trials.
5. Materials and methods
5.1. Materials
Solvents were purchased from Aldrich or Fischer and weresubjected to purification before use. Dioxanone (DX) and methyldioxanone (MeDX) were synthesized according to procedures pre-viously reported [41]. Tin (II) octanoate was used as received fromAldrich. Methoxyhydroxy poly(ethylene glycol), MPEG, of molarmass 2000, was used as received from International Laboratory,USA. Jeffamine® ED-2003 (Mw = 2000 g mol−1) was used as received
from Huntsman Corporation, USA. Dialysis membrane of MWCO3500 was purchased from Spectra® Pro, Spectrum Laboratories,Inc., USA. Phosphate buffered saline tablets (pH 7.4) were obtainedfrom Sigma–Aldrich.
R. Jeetah et al. / Mutation Research 768 (2014) 47–59 55
Table 6Cytotoxicity of drug-free and drug-loaded PEGylated nanomicelles toward some cell lines.
Copolymer Tumor model Results Ref.
DoxorubicinMPEG-PCL MCF-7 Minimum DOX concentration of 1 �M required in either
drug-free or drug-encapsulated copolymer after 5 daysincubation for almost 100% cytotoxic effect
Shuai et al. [37]
PEG-PCL-PEG MCF-7 Cell viability decreased to 13.2% within 48 h with DOX-loadedmicelle (DOX concentration: 10 �g/ml)
Cuong et al. [38]
PEG-PCL Adriamycin-resistant K562 Greater cytotoxicity of drug-loaded micelle (DOXconcentration: 6–12 �g/ml) than doxorubicin solution andability to reverse multidrug resistance
Diao et al. [29]
PLA-PEG-polyHis MCF-7 With DOX-loaded micelle, cell viability decreased withdecreasing pH
Lee et al. [33]
PaclitaxelPEG-PCL-PEG HepG2 and EMT6 HepG2 cells showed less sensitivity than EMT6 at the same
PTX concentrationZhang et al. [32]
MPEG-PLA-PTX conjugate H7402 At same PTX concentration of 20 ng/ml, same cell viability forpure drug and conjugate after 56 h incubationIncreasing PTX concentration in conjugate beyond 20 ng/mldoes not further decrease cell viability
Zhang et al. [39]
MPEG-PCL HEK293, L929 and 4T1 Drug-free micelle killed 50% of HEK 293 and L929 cells atconcentration of 3 mg/mlEquivalent cytotoxic activity by PTX-loaded micelle and freePTX against 4T1 cells
Wang et al. [40]
PEG-P(CL-co-TMC) HeLa After 24 h, the cell viability was more inhibited by PTX-loadedmicelles than by Taxol at 12.5 �g/ml of PTX
Danhier et al. [36]
CamptothecinPEG-SS-PBLG and PEG-PBLG SCC7 Cytotoxicity of CPT-loaded PEG-SS-PBLG was greater than that
of CPT-loaded PEG-PBLG micellesThambi et al. [35]
MPEG-PAE MDA-MB-231 Drug-loaded PMs at same CPT concentration showedsignificant cytotoxicity at pH 6.4 and less cytotoxicity at pH 7.4
Min et al. [34]
Cell line abbreviations: HeLa, human cervix epithelial carcinoma cells; HepG2, human liver carcinoma cells; MCF-7, breast carcinoma cells; L929, mouse fibroblast cells;MDA-MB-231, breast adenocarcinoma; SCC7, murine squamous cell carcinoma cells; H7402, human liver cancer cells; K562, human erythroleukemia tumor cells; EMT6,m rine bl
5
tRA
5
ca
5
a1
TP
ouse mammary carcinoma cells; HEK293, human embryonic kidney cells; 4T1, mu-glutamate).
.2. Drugs
Paclitaxel (98%), doxorubicin hydrochloride (>98%) and camp-othecin (>99%) were purchased from AK Scientific, Inc., USA,ichem International Enterprise Co. Limited, China and fromlomone Labs, Israel respectively.
.3. Copolymer synthesis
A range of diblock MPEG-b-P(DX-co-MeDX) and triblock P(DX-o-MeDX)-b-PEG-b-P(DX-co-MeDX) copolymers were synthesizeds described in our earlier publications [42,43].
.4. Loading of doxorubicin (DOX)
Doxorubicin hydrochloride (1 mg) was first neutralized bydding TEA (10 �L) in THF (2 ml) and the mixture was stirred for
h. The resulting solution was added to copolymer (10 mg) and
able 7olymeric micelles at various stages of clinical trials.
Polymeric micelles Block copolymer Drug Diameter (nm)
NK012 PEG-b-Pglu SN-38 20
NK105 MPEG-b-Pasp Paclitaxel 85
NK911 MPEG-b-Pasp Doxorubicin 15–60
NC 6004 PEG-b-Pglu Cisplatin 30
Genexol PM MPEG-b-P(DL-lactide) Paclitaxel 20–50
reast cancer cells; PAE, poly(�-amino ester); PA, palmitic acid; PBLG, poly(�-benzyl
distilled water (10 ml) under stirring. The solution was dialyzedagainst distilled water overnight (MWCO 3500 Da, Spectrum Labo-ratories, U.S.) [38].
5.5. Loading of paclitaxel (PTX)
Copolymer (10 mg) and paclitaxel (1 ml) were dissolved in ace-tone (2 ml) and distilled water (10 ml) was added under stirring.To remove the non-encapsulated drug, the resulting solution wasdialyzed against distilled water overnight [39].
5.6. Loading of camptothecin (CPT)
Copolymer (10 mg) and camptothecin (1 mg) were dissolvedin dichloromethane/methanol (9:1, v/v, 5 ml) and distilled water(10 ml) was added under stirring. The resulting solution was dia-lyzed overnight against distilled water [44].
Indication Clinical phase
Breast cancer IIAdvanced stomach cancer IIMetastatic pancreatic cancer IISolid tumorsBreast cancerPancreatic cancer
I/IIIV
Non-small-cell lung cancer in combination withcarboplatin
I/II
Pancreatic cancer in combination with gemcitabineOvarian cancer in combination with carboplatin
56 R. Jeetah et al. / Mutation Research 768 (2014) 47–59
and tr
5
ta
3ag0b
%
5
nnaa1tette
5
eww
6
t
three drugs were chosen from three classes of anticancer drugs
Fig. 11. Synthesis of diblock MPEG-b-P(DX-co-MeDX)
.7. Drug loading content
Calibration curves of the drugs were first drawn from UV spec-roscopy. Drug solutions of various concentrations were preparednd the absorptions of the solutions were measured by UV.
The peak at 480 nm for doxorubicin, 227 nm for paclitaxel and70 nm for camptothecin were used to draw the calibration curvesnd estimate the percentage of drug encapsulated. Straight lineraphs were obtained, with R2 values varying between 0.9909 and.9979. The drug loading content of nanoparticles was determinedy the digestion method and calculated using Eq. (1) [45].
Drug loading = weight of drug in nanoparticlesweight of nanoparticles
× 100 (1)
.8. In vitro drug release
Drug release was evaluated using the equilibrium dialysis tech-ique at 37 ◦C. A known amount of freeze-dried drug-loadedanomicelles was suspended in PBS solution (5 ml) (0.1 M, pH 7.4)nd was transferred to a dialysis bag. The dialysis bag was sealednd immersed in 100 ml of PBS. At predetermined time intervals,
ml of PBS solution was withdrawn from the external medium andhe drug content was quantified spectrophotometrically by UV. Thexternal medium was replenished with 1 ml of fresh PBS main-ained at 37 ◦C. By comparing the amount of the released drug andotal drug loading, cumulative releases were obtained. Each releasexperiment was performed three times.
.9. Statistical analysis
In all cases, experiments were carried out in triplicate and dataxpressed as mean ± standard deviation. Statistical comparisonsere performed using the Student’s t-test. P values less than 0.05ere considered statistically significant in all cases.
. Results and discussion
Most of the amphiphilic block copolymer micelles reported inhe literature consist of PEG and a biodegradable polyester (PLA,
iblock P(DX-co-MeDX)-b-PEG-b-P(DX-co-MeDX) [42].
PLGA, PCL) or polypeptide [poly(benzyl aspartate)]. The novelty ofour work resides in the use of diblock PEG-b-poly(ester-ether) ortriblock poly(ester-ether)-PEG-poly(ester-ether) nanomicelles fordrug encapsulation and release, where the hydrophobic polymerblock is a random copolymer of dioxanone and methyldioxanone[poly(dioxanone-co-methyl dioxanone)] (Fig. 11). The interest hereis that depending on the percentage of methyl dioxanone units dis-tributed along the chain, the overall crystallinity of the hydrophobicmicellar core is altered. This property can be used to controldegradation and consequently drug release to fit the drug ther-apeutic window [41,42]. Recently, the macromolecular structureand microstructure of PEG-PLA copolymers was shown to influencemicelle formation and cellular uptake [46].
We reported previously on the cytotoxicity and hemocom-patibility studies of the PEG-b-poly(ester-ether) micelles [43]. Nohemolysis and no erythrocyte agglomeration were noted for drug-free triblock copolymer nanomicelles in the concentration range0.125–1 mg/ml. The copolymer nanomicelles may therefore act assafe carriers of drugs via the intravenous route. The toxicity ofthe drug-free triblock copolymer nanomicelles, P(DX-co-MeDX)-b-PEG-b-P(DX-co-MeDX), %DX:%MeDX = 43:57 were tested over aconcentration range of 50–500 �g/ml via brine shrimp lethalitybioassay over 24 h. The copolymer micelles were highly biocom-patible and non-toxic at clinical administration limits with anLD50 concentration of ≈250 �g/ml and hence can be administeredintravenously at a total copolymer dose of 1000 mg taking 5 L(adult blood volume) as the apparent volume of distribution intoaccount.
6.1. Nanomicelles for doxorubicin, paclitaxel and camptothecindelivery
6.1.1. Drug propertiesFor the purpose of this study, three anti-cancer drugs have
been used namely doxorubicin, paclitaxel and camptothecin. These
namely: anthracyclines (doxorubicin); taxanes (paclitaxel), andalkaloids (camptothecin). Nanomicelle-encapsulation is expectedto enhance the bioavailability as well as half-life of the drugs. Theircharacteristics are summarized in Table 8.
R. Jeetah et al. / Mutation Research 768 (2014) 47–59 57
Table 8Characteristics of anti-cancer drugs used.
Drug pKa Water solubility (g/L) at 25 ◦C Log P* Molecular weight Half-life
Doxorubicin 8.94 to 9.53 Soluble 1.27 543.5 5 min [5]Paclitaxel −1 to 10.36 Insoluble 3 853.9 3 h to approx. 50 h
(Dose-dependent) [47]Camptothecin 3.07 to 11.71 0.511 1.74 348.4 5 min [48]
nt between n-octanol and water and is a well-established measure of the compound’sh
6
httvgea
rticewlwec
6
t3P
cDc5diMth
FMa
Fig. 13. Release profiles of paclitaxel-loaded P(DX-co-MeDX)-PEG-P(DX-co-MeDX)
* Log P: the log P value of a compound is the logarithm of its partition coefficieydrophilicity.
.1.2. Drug loadingCopolymers with variable lengths of hydrophobic and
ydrophilic blocks have been loaded with doxorubicin, pacli-axel and camptothecin anti-cancer drugs yielding micelles inhe range 130–300 nm (Table 9). Drug encapsulation efficienciesaried between 15% and 70%. The drug binding constants, whichive a good insight into drug encapsulation and release, werevaluated from UV spectroscopy as we reported previously fornti-TB drugs [42]. Some preliminary results are here presented.
As previously observed, the size of drug-free micelles was in theange of 130–160 nm and upon encapsulation of anti-cancer drugs,here was a general increase in size of micelles. There is no signif-cant difference between the encapsulation efficiencies of diblockopolymer micelles and that of triblock copolymer micelles. Thencapsulation efficiency of PTX and CPT did not change significantlyith copolymer composition unlike that of DOX. The encapsu-
ation efficiency of DOX into the copolymer micelles decreasedith increasing percentage of hydrophobic MeDX. This could be
xplained by an increased hydrophilicity of DOX even when neutralompared to PTX and CPT [49,50].
.1.3. Drug releaseThe drug release profiles of doxorubicin, paclitaxel and camp-
othecin from triblock copolymer micelles were recorded in PBS at7 ◦C. None of the release profiles show a burst effect (Figs. 12–14).TX, DOX and CPT all showed a release of 50–60% after 5 days.
There is a significant difference in the release kinetics betweenopolymer micelles with and without MeDX. For instance, 50% ofOX was released as follows: PDX-b-PEG-b-PDX: 33 h and P(DX-o-MeDX)-PEG-P(DX-co-MeDX): 46–69 h (%MeDX between 8% and7%) (Fig. 12). Diffusion of the drug from the hydrophobic core andegradation of the hydrophobic polymer segments are the dom-
nant mechanisms for drug release. The faster degradation of theeDX-containing copolymers is accounted for by an increase in
he amorphous character of the micelle core resulting in enhancedydrolysis and greater diffusion of the drug. The copolymer with
ig. 12. Release profiles of doxorubicin-loaded P(DX-co-MeDX)-PEG-P(DX-co-eDX) copolymer micelles, %DX:%MeDX = 100:0, 92:8 and 43:57 in PBS (pH = 7.4)
s a sink solution at 37 ◦C. Each value represents the mean ± S.D. (n = 3). P < 0.05.
copolymer micelles, %DX:%MeDX = 100:0, 92:8 and 43:57 in PBS (pH = 7.4) as a sinksolution at 37 ◦C. Each value represents the mean ± S.D. (n = 3). P < 0.05.
the highest MeDX content showed the fastest release rate irrespec-tive of the drug or the encapsulation efficiency.
In general, release of drug was fastest for doxorubicin, followedby camptothecin and paclitaxel as can be seen from Fig. 15. Thiscould be explained by the capacity of the drug to bind with themicellar core. It is expected that the stronger the drug-polymerinteraction, the higher would be the binding constant and theslower would be its release from the micelle. The binding constantswere calculated from Benesi–Hildebrand plots and are listed inTable 10. The binding constant of paclitaxel is about 25 times higherthan that of doxorubicin and 5 times larger than that of camp-tothecin.
6.2. Drug release kinetics
The zero-order kinetic and first order Higuchi models (Eqs. (2)and (3)) were applied to the release data up to the first 50% drug
Fig. 14. Release profiles of camptothecin-loaded P(DX-co-MeDX)-PEG-P(DX-co-MeDX) copolymer micelles, %DX:%MeDX = 100:0, 92:8 and 43:57 in PBS (pH = 7.4)as a sink solution at 37 ◦C. Each value represents the mean ± S.D. (n = 3). P < 0.05.
58 R. Jeetah et al. / Mutation Research 768 (2014) 47–59
Table 9Encapsulation of paclitaxel (PTX), doxorubicin (DOX) and camptothecin (CPT) in diblock and triblock copolymer micelles.
% DX:MeDX SF (nm) SL (nm) EE (%)
DOX PTX CPT DOX PTX CPT
MPEG-b-P(DX-co-MeDX)100:0 142 ± 3 230 ± 4 282 ± 3 159 ± 1 56 ± 0.56 54 ± 1.15 49 ± 1.5370:30 135 ± 3 245 ± 2 262 ± 3 168 ± 1 12 ± 0.58 57 ± 1.53 57 ± 0.5843:57 129 ± 4 234 ± 5 256 ± 2 147 ± 2 16 ± 0.70 59 ± 0.58 64 ± 0.70
P(DX-co-MeDX)-PEG-P(DX-co-MeDX)100:0 127 ± 5 189 ± 4 221 ± 5 139 ± 3 65 ± 1.14 54 ± 1.34 63 ± 0.8692:8 130 ± 3 177 ± 4 229 ± 4 167 ± 2 62 ± 0.69 53 ± 0.27 56 ± 2.3343:57 154 ± 3 300 ± 6 273 ± 6
EE, encapsulation efficiency; SF, diameter of drug-free micelle; SF, diameter of drug-load
Fig. 15. Release profiles of doxorubicin, paclitaxel and camptothecin-loaded P(DX-c(P
rR
M
M
woH
fckf
TCc
M
o-MeDX)-PEG-P(DX-co-MeDX) copolymer micelles, %DX:%MeDX = 92:8 in PBSpH = 7.4) as a sink solution at 37 ◦C. Each value represents the mean ± S.D. (n = 3).
< 0.05.
elease to analyze the drug release kinetics. Correlation coefficient2 was chosen to define the approximation accuracy.
t = M0 + K0t (2)
t = KHt1/2 (3)
here Mt, amount of drug dissolved in time t; M0, initial amountf drug in the solution; k0, zero-order release constant and KH,iguchi dissolution constant.
In the case of both diblock and triblock copolymer micelles and2
or all three drugs, namely DOX, PTX and CPT, the R values indi-ated that our systems are closer to a first-order sustained releaseinetics than a zero-order kinetics. For example, the R2 valuesor PTX-loaded P(DX-co-MeDX)-PEG-P(DX-co-MeDX) copolymer
able 10haracteristics of free and encapsulated paclitaxel (PTX), doxorubicin (DOX) andamptothecin (CPT).
Drug characteristics PTX DOX CPT
Molar volume (cm3)a 570.4 295.8 228.0Hydrogen bonding numbera 19 15 7Molar absorptivity of free drug
(M−1 cm−1)b26,200 11,500 27,350
Binding constant (M−1)c 5 × 106 2 × 105 1.1 × 106
Molar absorptivity ofencapsulated drug(M−1 cm−1)b
26,206 11,517 27,359
a Determined by Molecular Modeling Pro (ChemSW)b Determined by UV.c Determined by Benesi–Hildebrand plot, binding of drug to triblock P(DX-co-eDX)-b-PEG-b-P(DX-co-MeDX), %DX:%MeDX = 92:8 copolymer.
151 ± 3 33 ± 0.95 53 ± 0.81 52 ± 1.14
ed micelle.
micelles, %DX:%MeDX = 43:57 were 0.7549 and 0.9671 for the zero-order model and Higuchi model, respectively.
7. Conclusion
The best cure to cancer remains prevention. However, cancertherapeutics play and will continue to play a major role in cancertreatment. In spite of huge efforts to develop new anti-cancer drugs,only a few have reached commercialization stage. The new targeteddrugs or combined therapies have proved to be more efficient butthe problem of toxicity remains unsolved not withstanding multi-drug resistance (MDR). As reviewed in this paper, the developmentof nano-based systems has definitely proven to be more efficient interms of decreased toxicity, bioavailability and sustained release ofdrugs and combating MDR. Polymer design is key to the elaborationof effective drug carriers.
In that respect, we have tailored amphiphilic block copolymermicelles, PEG-b-P(DX-co-MeDX) and shown that they could beinteresting carriers for the delivery of anti-cancer drugs becauseof the ability to control drug release through tuning of thehydrolytic degradability of the micellar core. We reported pre-viously on the non-hemotoxicity of the drug free polymericmicelles. The encapsulation efficiency varied in the order pacli-taxel > camptothecin > doxorubicin. The binding of the drugs to thehydrophobic polymer core and the tuning of the latter’s hydrolyticdegradability through variation of the methyldioxanone units arefactors that control release of the drugs. The three drugs showedsustained release profile over a period of 5 days. The release of thedrugs varied in the order doxorubicin > camptothecin > paclitaxeland the copolymer with the highest MeDX content showed thefastest release. Release kinetics revealed that our systems followa first order sustained release.
We are of the opinion that micellar nanomedicine that candeliver multiple agents, i.e. combination therapy will be an impor-tant development in cancer therapeutics. Such nano-formulationswould allow sequential release of drugs within the required ther-apeutic window and could also be engineered as to target severalkey cancerous pathways.
Conflict of interest
None declared.
Acknowledgments
We thank the Tertiary Education Commission (Mauritius) fora PhD scholarship to R.J. and the Mauritius Research Council
(Mauritius) for supporting drug delivery research at the ANDI Cen-tre of Excellence for Biomedical and Biomaterials Research (CBBR).We thank the organizers of the IAMBR Meeting on Cancer Presentand Future Perspectives held at SSR Medical College (Mauritius)
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[riers for simultaneous encapsulation of synergistic actives, J. Am. Chem. Soc.
R. Jeetah et al. / Mutatio
n 16–18 September 2013 for inviting us to present our findings.e are also most indebted to our international collaborator, Prof.
iness Pillay, at the University of Witwatersrand, South Africa foris continuous support.
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