Curcumin-loaded Monomethoxy Poly(Ethylene Glycol)- Poly (-caprolactone) (MPEG-PCL)

Micelles
Curcumin is well known as efficient and safe anticancer agent. The problem with using curcumin is that it
is poorly soluble in water and is easily degraded by the body, resulting in low bioavailability. Thus,
curcumin cannot be used via the intravenous route, where it could possibly exert a maximal
pharmacological effect. mPEGPCL was employed in this study to prepare micelles. To overcome the
poor water-solubility of curcumin, curcumin was encapsulated into MPEG-PCL micelles by a self-
assembly method, producing curcumin /MPEG-PCL micelles. Amphiphilic nature of mPEG PCL with
hydrophilic PEG and hydrophobic PCL blocks provides an opportunity to form micelles in water. This
behavior can be explained as a consequence of copolymer self-assembly into micellar structure because
of its amphiphilic nature which, subsequently forces the hydrophilic PEG segments to serve as
hydrophilic shell and the hydrophobic PCL segments to become the micellar core.

the encapsulation efficiency was very high (>97%). The drug loading, particle size and PDI increased
with increase in curcumin/MPEG-PCL mass ratio in the feed. When the curcumin/MPEG-PCL mass ratio
in the feed was 20/100, the obtained Cur/MPEG-PCL micelles were not stable invitro (micelles tended
to aggregate, forming aggregates); thus, we chose 15/100 as the curcumin/MPEG-PCL mass ratio in feed
in our following protocol.

Fig 3.a the particle size distribution spectrum of freshly prepared Cur/MPEG-PCL micelles were
presented; this indicated that Cur/MPEG-PCL micelles had a very narrow particle size distribution. After
Cur/ MPEG-PCL micelles were freeze-dried, the re-solubility of these lyophilized Cur/MPEG-PCL
micelles was examined. As shown in Fig. 3c, these re-dissolved micelles were monodisperse (PDI
0.098 0.013) with mean particle size of 28.11.1 nm. In Fig. 3d, the TEM image of re-dissolved
micelles indicated that these micelles were still spherical and monodisperse with a mean diameter of ~23
nm. Thus, the freeze-dried Cur/MPEG-PCL micelle powder has good re-solubility.
X-ray diffraction peaks of curcumin dispersed in the XRD spectrum of Cur/MPEG-PCL micelles; this
implies that there are no curcumin crystals in the Cur/MPEG-PCL micelles. In Fig. 4b, the DSC curves
are shown. No melting transition peaks of curcumin are present, also confirming that there are no
curcumin crystals

Fig. 5 suggests that curcumin was molecularly incorporated in MPEG-PCL micelles simulated the
conformation of MPEG and PCL, and evaluated the bind affinity between curcumin and PEG or PCL.
The simulated structures of MPEG and PCL are shown in Fig. 5a and Fig. 5b. According to the docking
study, the binding affinity for PCL and curcumin was -17.99 kJ mol -1, while the value for PEG and
curcumin was -12.97 kJ mol_1; this indicates that the bind affinity between PCL and curcumin is stronger
than that between PEG and curcumin. This suggests that the curcumin is mainly located in the PCL core
of the MPEG-PCL micelles. curcumin could not be solubilized in PEG aqueous solution also implying
that curcumin is mainly located in the PCL core of MPEG-PCL micelles, rather than the PEG shell.

One of the major purposes of the encapsulation of curcumin in MPEG-PCL micelles is making curcumin
completely dispersible in aqueous media. The appearance of Cur/MPEGPCL micelles aqueous solution is
shown in Fig. 6. Curcumin cannot be dissolved in pure water (Fig. 6a) or 5% PEG solution (Fig. 6b), as
confirmed by the observation of a turbid yellow slurry. In contrast, Cur/MPEG-PCL micelle solution with
an equivalent quantity of curcumin was transparent (Fig. 6c), indicating full dispersiblity in water. Freeze-
dried Cur/MPEG-PCL micelles (Fig. 6d) were also fully dispersible in water (Fig. 6e). Thus, it is clear
that encapsulation of curcumin in MPEG-PCL micelles renders curcumin completely dispersible in
aqueous media, making curcumin intravenously injectable.
Results indicate that Cur/MPEG-PCL micelles can slow-release curcumin in vitro (Fig. 7a, about 54.6%
of total curcumin was released in 9 days)

Another reason for preparing Cur/MPEG-PCL micelles was to improve the pharmacokinetics of curcumin
in vivo. Blood was collected at different time intervals. For the MPEG-PCL micelleencapsulated
curcumin, the tmax, t1/2, AUC(0/t), AUC(0/N) and Cmax was 5 min, 34.2 min, 47642.1 mg L1 min1 ,
47864.6 mg L1 F min1 and 430.5 mg mL1 , respectively. For the free curcumin, the tmax, t1/2, AUC(0/t),
AUC(0/N) and Cmax were 5 min, 19.6 min, 7933.2 mg L1 min1 , 7944.6 mg L1 min1 and 305.7 mg mL1
, respectively. Thus, it was suggested that encapsulation of curcumin in MPEG-PCL micelles improved
the t1/2, AUC(0/t), AUC(0/N) and Cmax of curcumin in vivo.
Inhibition of angiogenesis

Angiogenesis is necessary for tumor growth, making inhibition of vessel formation an excellent target for
cancer therapy

To assess the functional integrity of the vasculature of each embryo, microangiography analysis was
performed on embryos. In Fig. 8, either free curcumin (Fig. 8b) or Cur/MPEG-PCL micelles (Fig. 8c)
treated embryos showed defective vascular formation in varying extents of severity; these intersegmental
vessels either sprouted abnormally or failed to form (arrows) compared with that of control embryo (Fig.
8a). This suggests that both free curcumin and Cur/MPEG-PCL micelles can inhibit the angiogenesis on
zebrafish model. Moreover, the inhibition of angiogenesis caused by Cur/ MPEG-PCL micelles was
confirmed in alginate-encapsulated tumor cells assay. In Fig. 9ad, alginate implant angiogenesis can be
directly observed. The vascularization of bead with Cur/ MPEG-PCL micelles treatment was clearly
suppressed compared with that of other treatments. The alginate implant angiogenesis also was quantified
by measuring the uptake of FITC-dextran into beads. FITC-dextran uptake was significantly lower in
mice treated with Cur/MPEG-PCL micelles compared with control groups (Fig. 9e). Meanwhile, MPEG-
PCL micelleencapsulated curcumin more efficiently induced the inhibition of angiogenesis than free
curcumin; this result may be caused by the improved pharmacokinetics of Cur/MPEG-PCL micelles
Anticancer activity in vitro and in vivo

The cytotoxicity of Cur/MPEG-PCL micelles to C-26 colon carcinoma cells was studied in vitro; results
are presented in Fig. 10a. Both curcumin and Cur/MPEG-PCL micelles efficiently killed C-26 colon
carcinoma cells. The IC50 for curcumin was 3.95 mg mL -1 , and that for Cur/MPEG-PCL micelles was
5.78 mg mL-1 . Compared to free curcumin, Cur/ MPEG-PCL micelles had slightly lower cytotoxicity,
which may be due to the slow release of curcumin.

The ability of Cur/MPEG-PCL micelles to inhibit the growth of C-26 colon carcinoma in vivo was
evaluated on a mouse model. Four groups of mice bearing C-26 colon carcinoma were intravenously
administered with normal saline, MPEG-PCL micelles (168 mg kg1 ), free curcumin (25 mg kg1 ) and
Cur/MPEG-PCL micelles (curcumin: 25 mg kg1 ), respectively. The tumor growth curves of each group
are presented in Fig. 10b. Results indicated that Cur/MPEG-PCL micelles treatment resulted in smaller
tumor volume compared to others treatment (p < 0.01, vs. control; p < 0.01, vs. MPEG-PCL; p < 0.05, vs.
curcumin). A representative tumor in each group is presented in Fig. 10c.

Tumor treated with Cur/MPEG-PCL micelles is smaller than that of the other groups. This in vivo study
indicates that: 1) systemic application of Cur/MPEG-PCL micelles (25 mg kg1 ) can inhibit the growth of
subcutaneous C-26 colon carcinoma in vivo; and 2) encapsulation of curcumin in MPEG-PCL micelles
enhances the anticancer activity of curcumin in vivo.

small size (27.3 1.3 nm), narrow size distribution (PDI 0.097 0.011), high encapsulation efficiency
(99.16 1.02%), high drug loading (12.95 0.15%), simple preparation method, and good re-solubility.

The encapsulation of curcumin in MPEGPCL micelles improved the t1/2 and AUC of curcumin (Fig. 7b).
The small size and hydrophilic PEG shell may allow Cur/MPEGPCL micelles to circulate for a long time
in vivo after systemic application. The long circulation time of MPEG-PCL micelles and the slow release
of curcumin in vivo may contribute to the improved t1/2 and AUC of curcumin.

The administration of Cur/MPEG-PCL micelles more efficiently inhibited the tumor angiogenesis than
that of free curcumin; this may due to the improved t1/2 and AUC of MPEG-PCL micelle-encapsulated
curcumin in vivo. Thus, i.v. application of Cur/MPEG-PCL micelles has potential in inhibiting tumor
angiogenesis.

The IC50 of free curcumin and Cur/ MPEG-PCL micelles was 3.95 mg mL1 and 5.78 mg mL1 ,
respectively. An in vivo study indicated that systemic application of Cur/MPEG-PCL micelles inhibited
the growth of subcutaneous C-26 colon carcinoma (p < 0.01). Moreover, Cur/MPEGPCL micelles
induced a stronger anticancer effect than free curcumin (p < 0.05); this implies that encapsulation of
curcumin in MPEG-PCL micelles enhanced the anticancer activity of curcumin in vivo. Curcumin has
cytotoxicity to cancer cells, thus it can directly kill cancer cells in vivo; meanwhile, curcumin can inhibit
angiogenesis, also resulting in suppression of tumor growth. Therefore, the anticancer effect of
Cur/MPEG-PCL micelles may be induced by inhibiting angiogenesis and directly killing cancer cells

Compared to previously reported nano-curcumin formulations, our Cur/MPEG-PCL micelles have

advantages such as small size, narrow size distribution, high encapsulation efficiency, high drug loading,
simple preparation method, and good re-solubility. Cur/MPEG-PCL micelles inhibited the growth of C-26
colon carcinoma through inhibiting angiogenesis and directly killing cancer cells. Encapsulation of
curcumin in MPEG-PCL micelles improved the pharmacokinetics, antiangiogenesis effect and anticancer
effect of curcumin in vivo.

pH sensitive

the percentage of curcumin released from the micelles slightly increased as the pH value decreased from
7.4 to 5.5. The sustained release of curcumin can be attributed to the entrapment of curcumin at the core
of micelles. Therefore, mPEGPCL copolymeric micelles can be regarded as highly attractive
nanocarriers for both time-controlled drug delivery for hydrophobic drugs and for the achievement of
different therapeutic objectives.
As a drug delivery system, mPEGPCL copolymer nanoparticles have some compensation, e.g: (1)
dipping the first-pass achieve and increasing bioavailability; (2) growing drug loading and encapsulation
competence; (3) dipping particle size and burst release while flourishing targeting; (4) avoid discovery
and elimination by the reticuloendothelial system, thus prolonging the circulation time of drugs in the
blood and civilizing steadiness; and (5) good security.

mPEG-PCL micelles give sustained/controlled release drug delivery system and targeted-drug delivery
method that might increase drug effectiveness and decrease drug struggle

Mechanism

mPEGPCL copolymer nanoparticles are largely dispersal- and deprivation-controlled release systems.
Hydrophobic drugs mostly are collected in the hydrophobic matrix. In dispersal-controlled systems, drugs
are dissolved or isolated in PCL polymers, and the release rate is restricted by drug diffusion in a PCL
matrix. The drugs neighboring the membrane surface can be released efficiently, while drugs seaside are
needed to mellow to the membrane surface first and then are released effectively. In the deprivation-
controlled system, drugs are dispersed in PCL, and the drug release time is assigned by degradation rate
outstanding to effect from PCL chain length, drug loading of nanoparticles, release means, and additional
factors. mPEGPCL nanoparticles with a slight sharing of particle size have a minor particle size than
PCL nanoparticles. Therefore, they can simply accumulate at inflammatory sites and then gradually
release the drugs. Specially, targeting molecules can be synthesized in the PEG end to increase energetic
targeting. Thus, the mixture of inactive and vigorous targeting can perform efficiently on abrasion
locations.

Composition weight of mPEG/PCL micelle

Physicochemical characterization of curcumin-loaded MePEG/PCL micelles. Curcumin was efficiently

loaded in different formulations of MePEG/PCL micelles (20:80, 40:60, 60:40 and 80:20)

From particle size measurement, we observed that with an increase in the molecular weight and
hydrophobic chain length (the PCL block) of the copolymer, the particle size increased from 68 1.3 nm
to 150 1.5 nm (Tab le 1). increase in molecular weight of copolymer increased the micelle size and
copolymer with large PCL blocks formed large micellar particles

small size of particles can be advantageous for passive targeting to tumor tissue due to an enhanced
permeability and retention effect

Hence, we can anticipate that our formulated micelle (which is smaller than 150 nm) will have an
enhanced circulation half-lives and reduced reticuloendothelial system uptake due to its nanoscale size
entrapment efficiency of curcumin in different micellar formulation was determined by HPLC. It was
evident that 20:80 and 40:60 MePEG/PCL micelles showed maximum entrapment efficiency (60.3 and
57.6% respectively, compared to other formulations) due to high molecular weight and large hydrophobic
chain length of the block copolymer in these micellar formulation compared with others

To assess the release profile of curcumin from the different MePEG/PCL micelles, studies were carried
out in vitro in PBS (0.01 M, pH = 7.4) over 7 days (Figure 3).

loading of the drug in nanosphere increases, the release rate decreases

As high entrapment was seen in 40:60 and 20:80 micellar formulations, this increases the hydrophobic
properties between curcumin within the nanosphere, favoring enhanced hydrophobichydrophobic
interaction of curcumin and PCL leading to a decreased drug release.

Further, it was observed that the release of curcumin from 20:80 micelles is slower as compared with
different formulation. This decrease of curcumin release rate could be due to the increased molecular
weight of the MePEG/PCL diblock copolymer

After observing the entrapment efficiency of curcumin in different formulations of MePEG/ PCL
micelles, the results revealed that the 20:80 and 40:60 MePEG/PCL micelles showed almost similar
entrapment efficiency however, 40:60 MePEG/PCL micelle was chosen as suitable formulation because
of its smaller size than the 20:80 formulations

Properties of curcumin were strongly influenced by solvent, water and pH

MePEG/ PCL micelles dramatically increased the stability of curcumin in PBS by protecting the
encapsulated curcumin against hydrolysis and biotransformation for a longer time.
Freeze dried formulations retained their physiochemical characteristics
thereby indicating their stability

REFERENCES
Danafar, Hossein. MPEGPCL copolymeric nanoparticles in drug delivery systems. Cogent Medicine
(2016), 3: 1142411
Danafar, Hossein et al. Biodegradable m-PEG/PCL Core-Shell Micelles: Preparation and
Characterization as a Sustained Release Formulation for Curcumin. Advanced Pharmaceutical
Bulletin, 2014, 4(Suppl 2), 501-510
Gou, MaLing, et al. Curcumin-loaded biodegradable polymeric micelles for colon cancer therapy in vitro
and in vivo. The Royal Society of Chemistry 2011 Journal. Nanoscale, 2011, 3, 1558-1567
Mohanty, Chandana, et al. Curcumin-encapsulated MePEG/PCL diblock copolymeric micelles: a novel
controlled delivery vehicle for cancer therapy. Nanomedicine (2010) 5(3), 433-449