Biodegradable block copolymer scaffolds for loading and delivering cisplatin anticancer drug

Article abstract of DOI:10.1002/zaac.201400030

A carboxylic acid substituted amphiphilic diblock copolymer scaffold with a hydrophilic PEG-chain and hydrophobic biodegradable poly(caprolactone) (PCL), which forms a cisplatin anticancer drug is reported herein. Cisplatin [cis-dichloro-diammine platinum(II), CDDP] was anchored on the polymer backbone through Pt-OOC-PCL chemical linkage that enabled self-assembly of the prodrug to produce 110±10 nm nanoparticles in water. These drug loaded nanoparticles were characterized by dynamic light scattering, electron microscopy, and X-ray diffraction. The polymer-drug conjugate burst instantaneously in saline and PBS to release 35% of the cisplatin drug for immediate administration. The remaining drug that retained in the polymer scaffold underwent slow and controlled release to deliver the drug over a period of 6-7 d. In the presence of esterase enzyme; the biodegradable PCL aliphatic ester backbone broke completely to release 100% loaded drugs within a few hours. This biodegradable diblock copolymer design strategy opens up new platform for cisplatin-polymer drug delivery approach. Copyright

Full text of DOI:10.1002/zaac.201400030

ARTICLE
DOI: 10.1002/zaac.201400030
Biodegradable Block Copolymer Scaffolds for Loading and Delivering
Cisplatin Anticancer Drug
Bapurao Surnar,[a] Pramod P. Subash,[a] and Manickam Jayakannan*[a]
Dedicated to Professor C. N. R. Rao on the Occasion of His 80th Birthday
Keywords: Block copolymers; Self-assembly; Prodrugs; Enzyme catalysis; Drug delivery
Abstract.
copolymer scaffold with a hydrophilic PEG-chain and hydrophobic for immediate administration. The remaining drug that retained in the
biodegradable poly(caprolactone) (PCL), which forms a cisplatin anti- polymer scaffold underwent slow and controlled release to deliver the
cancer drug is reported herein. Cisplatin [cis-dichloro-diammine plati- drug over a period of 6–7 d. In the presence of esterase enzyme; the
num(II), CDDP] was anchored on the polymer backbone through Pt– biodegradable PCL aliphatic ester backbone broke completely to re-
A carboxylic acid substituted amphiphilic diblock instantaneously in saline and PBS to release 35% of the cisplatin drug
OOC–PCL chemical linkage that enabled self-assembly of the prodrug lease 100% loaded drugs within a few hours. This biodegradable di-
to produce 110Ϯ10 nm nanoparticles in water. These drug loaded block copolymer design strategy opens up new platform for cisplatin-
nanoparticles were characterized by dynamic light scattering, electron polymer drug delivery approach.
microscopy, and X-ray diffraction. The polymer-drug conjugate burst
loaded drugs. This is also partially associated with the non-
biodegradability of the C–C bond in these acrylic or norbor-
nene polymers under in vivo (or in vitro) conditions. Thus,
new biodegradable polymeric scaffolds that are capable of de-
livering 100% cisplatin are very much in demand for cancer
treatment.
Introduction
Cisplatin is one of the widely employed inorganic anticancer
drugs for treating various types of cancers such as colon, lung,
ovarian, testicular, and so on.[1] Despite the clinical application
of cisplatin; neurotoxicity, non-selective accumulation of drugs
in healthy tissues, lack of receptor proteins on the cell mem-
Poly(caprolactone) (PCL) is one of the widely explored bio-
brane and low circulation time are some of the inherent limita-
degradable aliphatic polyester for various applications in bio-
tions associated with the its administration.[2] Further, more
medical industry.[11] Micellar assemblies of amphiphilic block
than 90% of the drugs were found to be rapidly cleared
copolymers based on polyethylene glycol-block-poly(caprolac-
through glomerular filtration which led to therapeutic ineffec-
tiveness in patients.[3] Recently, polymer based drug carriers
were explored for loading and delivering cisplatin drug to can-
cer tissues.[4] These polymer-cisplatin nanoparticles showed
enhanced accumulation in the intra-tumoral environment
through enhanced permeability and retention (EPR) effect.[5]
Poly(aspartic acid),[6] poly(glutamic acid),[7] poly(methacrylic
acid),[8] and amphiphilic block copolymers such as poly-(2-
hydroxy methacrylate),[9] poly [oligo-(ethyleneglycol)methyl-
methacrylate][9] and poly (oxanorborneyl anhydride)-b-poly-
[ω-oxanorborneyl-poly(ethyleneglycol)[10] are some of the im-
portant examples reported for cisplatin delivery. Most of these
polymer scaffolds were found to deliver about 50–60% of the
tone) (PEG-b-PCL) were reported as carriers for hydrophobic
drugs.[12] However, these biodegradable diblocks are not suit-
able for cisplatin conjugation due to the absence of functional
groups that are required for cisplatin chelation. Very recently,
pH-responsive carboxylic acid functionalized amphiphilic di-
blocks based on polyethylene glycol-block-poly (γ-carboxylic
caprolactone) (PEG-b-CPCL) have been reported.[13] These di-
block polymers were successfully demonstrated as oral drug
delivery vesicles for camptothecin and Ibuprofen under the
gastrointestinal tract.
In the presented investigation, the newly developed carbox-
ylic substituted diblock copolymers are employed as scaffolds
for anchoring and delivering cisplatin drugs. The design pro-
vides two advantages: (i) the PCL backbone has carboxylic
acid functionality for anchoring cisplatin, and (ii) the aliphatic
ester linkages in PCL can be completely degraded by esterase
enzyme under intracellular environment. Thus, the presented
biodegradable diblock copolymer design strategy is one of the
first examples for complete delivery of loaded cisplatin
(100%) for efficient cancer treatment. The schematic represen-
* Dr. M. Jayakannan
Fax: +91-20-2590 8186
E-Mail: jayakannan@iiserpune.ac.in
[a] Departmentof Chemistry
Indian Institute of Science Education and Research
Dr. Homi Bhabha Road
Pune 411008, Maharashtra, India
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/zaac.201400030 or from the au-
thor.
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tation of the approach is shown in Figure 1. The in-vitro re- Mn = 18,800 and Mw = 25,700 with narrow polydispersity
lease similar to physiological conditions confirmed the above (Mw/Mn = 1.36) (see SF-2, Supporting Information).
hypothesis. Thus, the custom designed carboxylic block co-
Cisplatin was treated with AgNO3 yielding the cis-diammi-
polymer architectures opens up new opportunities for con- nediaqua platinum(II) complex (see Scheme 1) and the AgCl
trolled delivery of cisplatin.
precipitate was removed by filtration.[15] Prior to complexation
with cisplatin drug, the block copolymer was converted into
its carboxylic sodium salt in double distilled water (pH = 6.8).
The aqua complex was stirred with polymer solution for 24 h
under dark (see Scheme 1). The resultant polymer-cisplatin
complex was filtered through 0.2 μm filters and dialyzed for
48 h to remove any insoluble particle, if present. The dialyzed
solution was lyophilized to yield grey colored polymer-cispla-
tin prodrug. The FT-IR spectra of nascent diblock polymer and
cisplatin conjugated polymer are shown in Figure 2. The carb-
onyl (–C=O) stretching peak appeared as discrete band at
1720 cm–1 in nascent polymer, which vanished and a new band
appeared at 1558 cm–1 with respect to (Pt–O–C=O) stretching
frequency of the metal carboxylate functional group.[16]
Additionally, a distinct peak at 545 cm–1 corresponding to
Pt–O (metal alkoxide) bond stretching was clearly visible in
the conjugate. These peaks matched with earlier reports and
confirmed the formation of polymer-cisplatin prodrug in the
present investigation.[16]
Figure 1. Biodegradable block copolymer approach for cisplatin drug
delivery.
Results and Discussion:
Synthesis and Characterization
The monomer was synthesized from commercially available
1,4-cyclohexanediol through multi-step reactions as reported
earlier.[13] It was subjected to ring opening polymerization
using polyethylene glycol monomethyl ether (MW 2000, about
45 repeating units) and Sn(Oct)2 as catalyst (as shown in
Scheme 1). The monomer to initiator ratio was maintained as
1
100 in the feed. H NMR spectroscopic analysis revealed the
presence of approx. 103 carboxylic units in the block copoly-
mers (see the Supporting Information for more details,
SF-1).[14] The GPC chromatograms showed mono-modal
distribution and the molecular weights were determined as
Figure 2. FT-IR spectra of the block copolymer (a) and the cisplatin-
polymer conjugate (b).
The drug loading content was estimated by thermo gravi-
metric analysis (TGA). The TGA plots for polymer, polymer-
Pt conjugate, and free cisplatin drug are shown in Figure 3.
The decomposition of the polymer started at 310 °C and it
completely degraded at 600 °C (Ͻ 1% remaining). Cisplatin
underwent stepwise decomposition and showed 60% weight
loss below 400 °C with respect to the loss of Cl and NH3 li-
gands. The platinum content remained unchanged up to
800 °C. In contrast, cisplatin-polymer conjugate showed the
combined decomposition profiles: (i) below 380 °C with re-
spect to the ligands (58%), (ii) between 380 to 580 °C with
respect to the polymers (24%), and (iii) with residual platinum
Scheme 1. Synthesis of the diblock copolymer and the polymer-cispla-
tin conjugate.
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Biodegradable Block Copolymer Scaffolds for Loading and Delivering Cisplatin Anticancer Drug
Figure 3. TGA plots of free cisplatin, the polymer, and the polymer-
cisplatin conjugate.
above at 600 °C (16% remaining). Following the procedure
reported by Xu et al. the drug conjugation efficiency (DCE)
was estimated using the equation:[10]
DCE = mPt,exp/mPt,theo ϫ100%
= (WPt/MPt)/ (Wacid/2Macid)ϫ100%
where mPt,theo is the theoretical molar amount of Pt; mPt,exp is
the experimental molar amount of Pt; WPt is the weight percent
of Pt measured by TGA; MPt is the molecular weight of Pt;
Wacid is the weight percent of acid repeating unit calculated by
TGA data; Macid is the molecular weight of acid repeating
unit.[10] Based on this equation, the DCE was obtained as
50.6% and the drug loading content (DLC) of polymer sample
was calculated as 16% (see Experimental Section for more
details).
Figure 4. DLS histogram (a); FE-SEM image (b); HR-TEM image (c);
selective area electron diffraction (SAED) pattern (d); powder WXRD
pattern (e); water contact angle (f) of polymer and polymer drug conju-
gate.
indicating their amorphous nature (see SF-3, Supporting Infor-
mation). The bulk sample was subjected to wide angle power
X-ray diffraction analysis (see Figure 4e). The polymer did not
Morphology of the Polymer-Cisplatin Conjugate
The amphiphilic diblock copolymer has hydrophilic PEG show any sharp peaks indicating their amorphous nature. On
chains and hydrophobic PCL core for self-organization in the other hand, sharp crystalline peaks appeared in the
water. The hydrophobic core was conjugated through Pt-OOC- polymer-drug conjugate with respect to 111, 200, 220, and
polymer linkage, which leads to the nanoparticle assembly 331 lattices (as observed in the SAED images, see Figure 4d).
having rigid core with flexible PEG tails as corona. The size These crystalline lattices were found to match with the cispla-
and shape of the polymer-drug conjugate was studied by dy- tin-data that were reported in the literature.[17] Further, water
namic light scattering and electron microscopes. Dynamic light contact angle (WCA) measurements were also carried out to
scattering (DLS) profiles of the drug conjugate showed a confirm the hydrophilic or hydrophobic nature of polymer and
monomodal distribution with average diameter of 120Ϯ10 nm their drug conjugates. WCA for the block polymers and its
(see Figure 4a). The FE-SEM image of the drug conjugate (see cisplatin conjugates were determined by the sessile drop tech-
Figure 4b) showed the existence of spherical objects with nique. The photographs of water droplets on the polymer film
average diameter of 110Ϯ10 nm. HR-TEM image of the drug and their corresponding WCA values are shown in Figure 4f.
conjugates also showed the spherical particle morphology with The diblock polymer exhibited WCA value of Ͻ1° due to its
average sizes of 120Ϯ10 nm (see Figure 4c). All these three complete water solubility and highly hydrophilic nature. The
independent techniques showed similar particle sizes, which WCA of the drug conjugated polymer were escalated to
confirmed the nano-particulate nature. The selective area elec- 30Ϯ5° (see Figure 4f). This revealed that the formation of
tron diffraction (SAED) pattern (see Figure 4d) showed highly Pt-OOC bond (see Scheme 1) enhanced the hydrophobicity of
crystalline states in the drug-polymer particles. Based on the the drug conjugate through cisplatin conjugation. Thus, based
GATAN software data base, these lattices were assigned to on these studies; it may be concluded that the diblock polymer
111, 200, 220, and 331 lattices.[17] On the other hand, the wrapped the cisplatin and stabilized them in aqueous medium
SAED pattern of the polymer did not show any diffraction as 110 nm size particles for drug administration.
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B. Surnar, P. P. Subash, M. Jayakannan
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Figure 5. Schematic representation of the cisplatin release from the polymer conjugate in in saline and its complexation reaction with
o-phenylenediamine (a); absorbance of the cisplatin-OPD conjugate at different time intervals (b) in PBS at 37 °C.
In vitro Drug Release Studies
The in vitro drug release profile of the polymer-Pt drug con-
jugate was investigated as shown in Figure 5a. The chloride
and phosphate anions in saline (aqueous NaCl, pH = 6.8) and
phosphate buffer saline (PBS, pH = 7.4), respectively, are
highly reactive to cisplatin-carboxylate linkages. The chloride
anions attach on the Pt-OOC-polymer linkage to regenerate the
cisplatin as drug during the in vitro studies (see Figure 5a).
The amount of cisplatin released in the media could be esti-
mated with the help of o-phenylenediamine colorimetric assay
(see Figure 5b).[9]
In this method, cisplatin released from the polymeric carrier
were treated with OPD (o-phenylenediamine) for the estima-
tion of the released drug. The amount of cisplatin drug released
in this solution could be determined with high accuracy by
measuring the absorbance at 706 nm (the absorbance spectra
of OPD-Pt complex). Absorbance spectra recorded for the re-
lease of the cisplatin in saline over a period of 4 d is shown
in Figure 5b. The absorbance spectrum of OPD treated aliquots
(see Figure 6c) exhibited an increase in the intensity with re-
lease time. The cumulative release was calculated as follows:
Cumulative release (in %) = Cn ϫVo / mϫ100%
where Cn is the amount of loaded cargo in the nth sample, Vo
is the total volume and m is total amount loaded in prodrug.
The cumulative release patterns for the conjugate drug in
the presence of double distilled water, saline, and PBS are
shown in Figure 6. In water, the polymer-drug adduct was
completely stable and practically no drug was released even
after 6 d. In saline, the chloride ion induced the de-chelation of
the polymer-drug conjugate, which typically occurred in two-
Figure 6. Cumulative drug release of cisplatin in water, saline and
PBS (a). Plots of log (Mt/Mϱ) vs. log t for stage-I (b) stage-II; (c) of
stages. An initial burst release of the drug-polymer carrier
occurred within 10 h with 34% release of cisplatin. Sub-
the cisplatin release in saline and PBS; schematic representation of the
sequently, the release became very slow and the remaining disassociation of polymer-cisplatin conjugate(d).
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Biodegradable Block Copolymer Scaffolds for Loading and Delivering Cisplatin Anticancer Drug
Table 1. Drug release kinetic parameters.
Condition employed
Stage I
N
Stage II
n
k
R2
k
R2
PBS
Saline
Esterase (initial)
Esterase (after 70 h)
0.27
0.21
0.32
0.39
0.625
0.689
0.125
0.821
0.982
0.981
0.978
0.986
0.95
1.01
–
0.1ϫ10–2
0.995
0.996
–
0.9ϫ10–2
–
–
a)
–
–
a) The first two phases are similar to that of PBS alone: Stage I with n = 0.29 and k = 0.628; Stage II with n = 0.9 and k = 0.11ϫ10–2.
20–30% drug was released over a period of 5–6 d. PBS also was not accessible in saline or PBS. A similar trend was earlier
showed a similar profile as noticed in the saline.
observed by others in acrylic and norbornene based polymer
The drug release kinetics in saline and PBS in both stages scaffolds.[10] This suggested that under normal conditions (in
were estimated by the Peppas model (power law).[18] The saline or PBS) only 67% of the cisplatin could be released
Peppas power law can be stated as Mt/Mϱ = ktn, where Mt and from the polymer scaffolds.
Mϱ are cumulative releases of drug at time t and infinite time
respectively, n is a release exponent, and k is the rate constant.
This equation can be written in linear form as
Mt
Enzymatic Release Studies
log ( ) = n log t + log k
Mϱ
The block copolymer scaffold has two parts: (i) biocompat-
ible and non-biodegradable hydrophilic PEG units, and (ii)
hydrophobic and biodegradable poly(caprolactone) units hav-
ing aliphatic ester linkages. The aliphatic ester linkages in the
long PCL backbone could be easily chopped under carboxylate
esterase enzyme. Since certain cancer cells such as liver and
colon have over expression of esterase enzyme;[21] the in vitro
analysis of the polymer-cisplatin drug conjugate in the pres-
ence of esterase enzyme may provide more insight into their
intracellular delivering capability.[22,23]
The diffusion exponent n and the kinetic constant k can be
estimated from the slope and the intercept of the plot of log
(Mt/Mϱ) vs. log t, respectively. According to the Peppas model
(if n Ͻ 0.43), the release of drug happened by both diffusion
and erosion processes. The value of n between 0.43 and 1 cor-
responds to the non-Fickian (anomalous control) in nature. The
value of n = 1 represents zero order drug release. This equation
is normally applicable for the initial 60% of the fractional
drug release or for the values in the interval of
0.1 Ͻ Mt/Mϱ Ͻ 0.7.
Cumulative release profiles of the polymer-drug conjugate
in the presence of 10 U of esterase are shown in the Figure 7a.
It is very clear from the plot that almost all 100% drug was
completely released in less than 10 h by esterase. To further
prove the role of esterase enzyme; a control experiment was
carried out in which the polymer-drug conjugate was initially
exposed to PBS for 72 h (3 d) and subsequently treated with
10 U esterase (see Figure 7a). It is very interesting to notice
that only 40% of the drug is released in PBS up to 72 h and
the remaining drug released immediately as soon as it was
exposed to esterase enzyme. A further Peppas model was ap-
plied to the release profiles of cisplatin from nanoparticles in
presence of esterase alone and PBS followed by esterase en-
zyme. The plots were fitted to above model (see Figure 7), and
n and k values are listed in Table 1. In the presence of esterase,
the cisplatin delivered in a single step with n value of 0.32.
This expresses the release in diffusion plus erosion pathway
(see Figure 7b). In the control experiment, it followed three
steps kinetics that includes two stages of PBS and a final ester-
ase triggered release. As expected, the drug release kinetics of
the initial two phases are similar to that of the PBS alone ex-
periment (see SF 4 for details, Supporting Information). The
esterase assisted drug release went along almost identical to
that of enzyme initially added experiment. (n = 0.39) (see Fig-
ure 7c). This difference was attributed to the higher penetrating
This model was widely employed for understanding the
drug release mechanism from various polymeric carriers such
as micelles, vesicles and nanogels and so on. Zhuang and co-
workers employed this model to study the drug release from
the nanogels.[19] Lecommandoux and et al. and Bapurao et al.
used the Peppas model for understanding the drug release from
nano-vesicular assemblies.[13,20] In this study, the biphasic re-
lease profiles of cisplatin in the presence of saline and PBS
were subjected to analysis based on the Peppas model. The
plots were fitted to the above equation and their kinetics plots
log (Mt/Mϱ) against log t are showed in Figure 6b and c. The
rate constant k and n values are provided in Table 1. The nano-
particles showed an initial burst release in stage 1 (0–9 h) with
low n values (n Ͻ 0.3) with respect to the 38% cisplatin re-
lease (Figure 6b). This was attributed to the effect of chloride
ion, which leads to the disassembly of nanoparticle and subse-
quent cisplatin release with both diffusion plus erosion path-
way. Stage II exhibited the sustained release with n values
(n ≈ 1), which indicates that the remaining 10–15% of cispla-
tin release followed zero order kinetics (Figure 6c). Hence,
these observations prove that cisplatin-prodrug show sustained
release with zero order kinetics, which may help to increase
the circulation time in blood plasma.
Based on these kinetic data, the schematic representation of
release pattern for cisplatin has been proposed in Figure 6d.
However, it was rather surprise that more than 40% of the Pt- ability of the enzyme on the nanoparticle, which is already
drugs were permanently bonded to the polymer scaffold that disrupted by the PBS prior to enzyme action.
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gradable block copolymer assemblies for loading and de-
livering cisplatin.
Conclusions
A biodegradable block copolymer structure was developed
with enzymatic cleavable polycaprolactone backbone and car-
boxylic acid functional groups for anchoring cisplatin drugs.
The polymer-drug conjugate appeared in the form of 110 nm
spherical particles, which could in principle be administered
selectively to the cancer tissue via EPR effect. The drug conju-
gate was very stable and can be stored in water for a long
period without any cleavage. The dispersion of the polymer-
drug conjugate in saline and PBS leads to the burst release of
35% cisplatin in the reservoir for immediate drug adminis-
tration. About 20–30% of the remaining drugs undergo slow
and controlled release over a period of 6–7 d. In the presence
of esterase enzyme, the biodegradable PCL aliphatic ester link-
age cleaved instantaneously and all the cargoes (100% of the
drugs) released. Thus, the multi-stimuli cleavable biodegrada-
ble block copolymer scaffold is a potential vector for cisplatin
delivery for cancer treatment.
Experimental Section
Materials: Tin(II) 2-ethylhexanoate [Sn(Oct)2], polyethylene glycol
monomethyl ether (MW = 2000, here after referred as PEG), cisplatin
and o-phenylenediamine(OPD) were purchased from Sigma Aldrich.
PEG and catalyst Sn(Oct)2 were dried under vacuum prior to use. All
other solvents like tetrahydrofuran (THF) and trifluoroacetic acid
(TFA) are purchased locally and distilled and kept in an inert atmo-
sphere prior to use. The monomer tert-butyl 3-[(7-oxooxepan-4-
Figure 7. Cumulative drug release (a); plots of log (Mt/Mϱ) vs. log t
for esterase (initial) (b); esterase after (70 h) (c); schematic representa-
tions of the dissociation of polymer-cisplatin conjugate in the presence yl)oxy] propanoate was synthesized as reported by us earlier.[13]
of esterase (d).
Measurements: NMR was recorded with a 400-MHz JEOL NMR
Spectrophotometer. All NMR spectra were recorded in CDCl3 contain-
ing TMS as internal Standard. Gel permeation chromatographic (GPC)
The presented investigation has demonstrated the role of two
analysis was performed with Viscotek VE 1122 pump, Viscotek VE
stimuli for cisplatin release based on PBS buffer (or saline)
3580 RI detector, and Viscotek VE 3210 UV/Vis detector in tetra-
and esterase enzyme. It was clear from the in vitro studies that
the polymer scaffold was biodegradable under esterase enzyme
hydrofuran (THF) using polystyrene as standards. Thermal stability of
the polymers was determined with a Perkin-Elmer thermal analyzer
and facilitated the complete drug release, which was not ac- STA 6000 model at a heating rate of 10 °C·min–1 in a nitrogen atmo-
sphere. Water contact angle measurements were performed with a
GBX model (DIGIDROP contact angle instrument) using windrop
software. Extreme care has been taken in carrying out sessile contact
angle measurements to monitor contact angle values within 1 min to
avoid the evaporation effects (repetition of the word contact angle).
All contact angle measurements were carried out at room temperature
(27 °C) under constant humidity (40–50%). The absorption spectra
were recorded with a Perkin-Elmer Lambda 45 UV/Visible spectro-
photometer. Dynamic light scattering (DLS) was done with a Nano ZS-
90 apparatus utilizing 633 nm red laser (at 90° angle) from Malvern
cessible under normal conditions (such as PBS or saline). In
general, the release profile of the drugs at intracellular level
was assisted by more than one stimuli such as blood plasma
(by chloride, phosphate, and other ions), enzymes (like ester-
ase) and so on. Thus, the biodegradable diblock copolymer
design, in principle, is capable of delivering 100% loaded cis-
platin drugs through the combination of more than one degrad-
able pathways.
The current investigation has clearly evident for the achieve-
ment of biodegradable diblock polymer assemblies for metal Instruments. FE-SEM images were recorded with a Zeiss Ultra Plus
scanning electron microscope. For FE-SEM analysis, the samples were
prepared by drop casting on silicon wafers and coated with gold. TEM
images were recorded with a Technai-300 instrument by drop casting
the sample on Formvar-coated copper grid. The fluorescent micro-
graphs were collected with a Carl Zeiss Axiovert 200 microscope.
based drugs such as cisplatin. The concept was successfully
demonstrated based on new diblock PEG-b-PCL design as
well delivering the drug by dual stimuli: saline and esterase
enzyme. However, the cytotoxicity of the polymer, polymer-
drug conjugates and their cellular uptake mechanism are fur-
ther need to be confirmed. Nevertheless, the current investiga-
Polymer Synthesis: Ring opening polymerization of tert-butyl 3-[(7-
tion provides the first time insight to the development of biode- oxooxepan-4-yl)oxy] propanoate was performed with [M0]/[I0] = 100
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Biodegradable Block Copolymer Scaffolds for Loading and Delivering Cisplatin Anticancer Drug
and polyethylene glycol monomethyl ether as initiator by following o-phenylenediamine (OPD) Colorimetric Assay: Samples with un-
our earlier report. PEG (77.5 mg, 0.0387 mmol) was taken in a flame
dried Schlenk tube and dry toluene (2.0 mL) was added in a nitrogen
atmosphere. To this mixture, Sn(Oct)2 (7.8 mg, 0.0193 mmol) was
added and the content was stirred at 25 °C for 15 min under nitrogen
known cisplatin (Pt) content were added to 0.5 mL of OPD solution
in N,N-dimethylformamide (DMF) (1.2 mg·mL–1) and heated for 2 h
at 100 °C. The amount of Pt present in the sample was determined by
measuring the absorbance at 706 nm (absorbance maxima of OPD-
purge. The monomer (1 g, 3.87 mmol) was added to the above mixture Pt complex). Molar extinction coefficient was calculated for OPD-Pt
and the polymerization mixture was stirred at 25 °C for 15 min under calculated as 24,310 L·mol–1·cm–1, alike to literature.[24] The concen-
nitrogen purge. The polymerization tube was immersed in preheated tration of Pt released from the conjugate was expressed as a ratio of
oil bath at 110 °C and the polymerization was continued for 48 h with the amount of platinum in the releasing solution from the polymer
constant stirring. The polymerization mixture was precipitated in
MeOH. The polymer was re-dissolved in THF and precipitated again
in methanol. The purification was done at least twice to obtain highly
pure polymer. Yield: 710 mg (71%). 1H NMR (400 MHz, CDCl3):
δ = 4.13 (s, 2 H, OCH2), 3.64 (m, 3.8 H, PEG and –OCH2–), 3.45 (s,
1 H, –CH–), 3.38 (s, 3 H, OCH3), 2.44 (t, 2 H, COCH2), 2.35 (t, 2 H,
COCH2), 1.93–1.81 (m, 2 H, –CH2–), 1.81 –1.67 [m, 4 H, CH2(CH2)],
1.44 (s, 9 H, tert-butyl) ppm. 13C NMR (100 MHz, CDCl3): δ = 173.63
(C=O), 170.81 (O=C–O), 80.72 [OCH(CH2)], 75.58 (OCH2), 70.68
(OCH2), 65.13 (COCH2), 61.47 (COCH2), 36.60 (CH2), 33.04 (CH2),
29.81(CH2), 28.86 (tert-butyl), 28.22. FT-IR: ν˜ = 2973, 2931, 1726
(C=O ester), 1457, 1364, 1251, 1156, 1156, 1099, 1062, 957, 898, 845,
757 cm–1. GPC molecular weights: Mn = 18,800, Mw = 25,700 and
Mw/Mn = 1.36.
backbone.
In vitro Drug Release Studies: Cisplatin loaded nanoparticles were
taken in a dialysis bag (in 3 mL) and they were immersed in a 100 mL
beaker and dialyzed at 37 °C with constant stirring. At specific time
intervals, 1.0 mL of the dialysate was withdrawn and replaced with an
equal volume of fresh buffer (or) saline. The amount of molecule (or
drug) released in each aliquot was measured using OPD colorimetric
assay by using absorption spectroscopy to quantify their percentage of
cumulative release. Cumulative release (%) = Cn ϫVo / mϫ100 where
Cn is the amount of loaded cargo in nth sample, Vo is the total volume
and m is the total amount loaded in vesicles. For esterase assisted
release studies 10 units of enzyme was used, above mentioned pro-
cedure was followed for calculation of cumulative release.
Supporting Information (see footnote on the first page of this article):
NMR spectra, GPC plots, drug loading content calculations, SAED
pattern of polymer are given.
Synthesis of Carboxylic Substituted Poly(Caprolactone) (PEG-b-
CPCL100): Trifluoroacetic acid (1 mL) was added slowly into PEG-b-
BuCPCL100 (500 mg) in dry DCM (15 mL) and the polymer solution
was stirred at 25 °C for 30 min. The solvents were evaporated and the
polymer was re-dissolved in THF and precipitated in cold methanol.
Acknowledgements
1
The purification was repeated at least twice to get pure polymer. H
NMR (400 MHz, CDCl3): δ = 4.13 (t, 2 H, CH2OH), 3.63 (m, 3.8 H,
PEG and OCH2), 3.55 (m, 1 H, OCH), 2.54 (t, 2 H, CH2COOH), 2.37
(t, 2 H, COCH2), 1.97–1.65 [m, 4 H, –OCH (CH2)2]. FT-IR: ν˜ = 3447,
2932, 2450, 1711(C=O acid), 1355, 1257, 1175, 1096, 1059,
955 cm–1.
The authors thankfully acknowledge research grants from Department
of Science and Technology (DST), New Delhi, INDIA, under nano-
mission initiative project SR/NM/NS-42/2009 and SERC Scheme pro-
ject SR/S1/OC-37/2013. Bapurao thanks CSIR, New Delhi, India for
research fellowship. Pramod thanks IISER-Pune for research fellow-
ship.
Preparation of Aquated Cisplatin [Pt(NH3)2(OH2)2]2+: For synthe-
sis of aquated cisplatin, 18 mg (0.059 mmol, 1 equiv.) of cisplatin was
partially dissolved in H2O (18.0 mL). To this mixture, silver nitrate
(20.3 mg, 0.119 mmol, 2 equiv.) was added and the resulting reaction
mixture was stirred at room temperature for 24 h. Formation of aquated
cisplatin confirmed by milky white colored silver chloride precipi-
tation. Silver chloride was removed by centrifuging at 10,000 rpm for
1 h. Finally, the aquated cisplatin was obtained by filtration through
0.2 μm filter.
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Received: January 17, 2014
Published Online: April 8, 2014
1126
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© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Z. Anorg. Allg. Chem. 2014, 1119–1126