Thermoresponsive Polymer Micelles as Potential Nanosized Cancerostatics

Article abstract of DOI:10.1021/acs.biomac.5b00764

An effective chemotherapy for neoplastic diseases requires the use of drugs that can reach the site of action at a therapeutically efficacious concentration and maintain it at a constant level over a sufficient period of time with minimal side effects. Currently, conjugates of high-molecular-weight hydrophilic polymers or biocompatible nanoparticles with stimuli-releasable anticancer drugs are considered to be some of the most promising systems capable of fulfilling these criteria. In this work, conjugates of thermoresponsive diblock copolymers with the covalently bound cancerostatic drug pirarubicin (PIR) were synthesized as a reversible micelle-forming drug delivery system combining the benefits of the above-mentioned carriers. The diblock copolymer carriers were composed of hydrophilic poly[N-(2-hydroxypropyl)methacrylamide]-based block containing a small amount (～5 mol %) of comonomer units with reactive hydrazide groups and a thermoresponsive poly[2-(2-methoxyethoxy)ethyl methacrylate] block. PIR was attached to the hydrophilic block of the copolymer through the pH-sensitive hydrazone bond designed to be stable in the bloodstream at pH 7.4 but to be degraded in an intratumoral/intracellular environment at pH 5-6. The temperature-induced conformation change of the thermoresponsive block (coil-globule transition), followed by self-assembly of the copolymer into a micellar structure, was controlled by the thermoresponsive block length and PIR content. The cytotoxicity and intracellular transport of the conjugates as well as the release of PIR from the conjugates inside the cells, followed by its accumulation in the cell nuclei, were evaluated in vitro using human colon adenocarcinoma (DLD-1) cell lines. It was demonstrated that the studied conjugates have a great potential to become efficacious in vivo pharmaceuticals. (Figure Presented).

Full text of DOI:10.1021/acs.biomac.5b00764

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Article
Thermo-responsive polymer micelles as potential nano-sized cancerostatics
Richard Laga, Olga Janoušková, Karel Ulbrich, Robert Pola, Jana
Blažková, Sergey K. Filippov, Tomas Etrych, and Michal Pechar
Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.5b00764 • Publication Date (Web): 08 Jul 2015
Downloaded from http://pubs.acs.org on July 12, 2015
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Thermoꢀresponsive polymer micelles as potential
nanoꢀsized cancerostatics
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Richard Laga*, Olga Janoušková, Karel Ulbrich, Robert Pola, Jana Blažková, Sergey K.
Filippov, Tomáš Etrych and Michal Pechar
Institute of Macromolecular Chemistry Academy of Sciences of the Czech Republic, v.v.i,
Department of Biomedical Polymers, Heyrovský sq. 2, 162 06 Prague, Czech Republic
Keywords
RAFT polymerization; polymer therapeutics; thermoꢀresponsive micelles; light scattering; laser
scanning confocal microscopy; cancer therapy
Abstract
An effective chemotherapy for neoplastic diseases requires the use of drugs that can reach the
site of action at a therapeutically efficacious concentration and maintain it at a constant level
over a sufficient period of time with minimal side effects. Currently, conjugates of highꢀ
molecularꢀweight hydrophilic polymers or biocompatible nanoparticles with stimuliꢀreleasable
antiꢀcancer drugs are considered to be some of the most promising systems capable of fulfilling
these criteria. In this work, conjugates of thermoꢀresponsive diꢀblock copolymers with the
covalently bound cancerostatic drug pirarubicin (PIR) were synthesized as a reversible micelleꢀ
forming drug delivery system combining the benefits of the above mentioned carriers. The diꢀ
block copolymer carriers were composed of hydrophilic poly[Nꢀ(2ꢀ
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hydroxypropyl)methacrylamide]ꢀbased block containing a small amount (~ 5 mol. %) of
comonomer units with reactive hydrazide groups and a thermoꢀresponsive poly[2ꢀ(2ꢀ
methoxyethoxy)ethyl methacrylate] block. PIR was attached to the hydrophilic block of the
copolymer through the pHꢀsensitive hydrazone bond designed to be stable in the blood stream at
pH 7.4 but to be degraded in the intratumoral/intracellular environment at pH 5 ꢀ 6. The
temperatureꢀinduced conformation change of the thermoꢀresponsive block (coilꢀglobule
transition), followed by selfꢀassembly of the copolymer into a micellar structure, was controlled
by the thermoꢀresponsive block length and PIR content. The cytotoxicity and intracellular
transport of the conjugates as well as the release of PIR from the conjugates inside the cells,
followed by its accumulation in the cell nuclei, were evaluated in vitro using human colon
adenocarcinoma (DLDꢀ1) cell lines. It was demonstrated that the studied conjugates have a great
potential to become efficacious in vivo pharmaceuticals.
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Introduction
Currently, neoplastic diseases, together with cardiovascular diseases, two of the most common
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fatal illnesses, represent a serious health problem in patient populations . The treatment of
neoplastic diseases using conventional chemotherapeutics – lowꢀmolecularꢀweight compounds
with cancerostatic effects – may lead to the suppression or even a complete cure of the disease;
however, such treatment is usually associated with particular side effects. These side effects are
mainly the result of ineffective localization of a cytotoxic drug in a tumor, often leading to the
irreversible damage to healthy cells and tissue. Moreover, the treatment using lowꢀmolecularꢀ
weight chemotherapeutics commonly requires relatively frequent dosing as the drug
concentration at the site of action rapidly decreases due to rapid blood clearance, glomerular
filtration and drug elimination.
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The chemotherapeutic strategy using conjugates of highꢀmolecularꢀweight (HMW) synthetic
hydrophilic polymer carriers with covalently linked drugs is considered to be one of the most
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effective ways to prolong blood clearance and reliably reduce unwanted side effects . Based on
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the differences in the endothelial permeability of the healthy and the malignant tissues, such
conjugates are able to deliver the drug preferentially to the tumor tissue without significantly
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affecting the healthy cells . Furthermore, properly chosen stimuliꢀsensitive biodegradable
linker inserted between the polymer backbone and the drug may ensure the conjugate stability in
the circulation and a longꢀlasting release of the drug directly inside the tumor or the cancer cells
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It was shown that the level of the passive accumulation of the conjugates in the solid tumors
due to the enhanced permeability and retention (EPR) effect depends to a large extent on the size
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or molecular weight of the polymer carrier . Current synthetic procedures enable the preparation
of polymerꢀdrug conjugates with a broad range of molecular weights and sizes of polymer coils
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Unfortunately, the elimination of such conjugates from the body after delivering the drug
(one of the basic requirements for drug delivery carriers) is limited by the molecular weight, e.g.,
for Nꢀ(2ꢀhydroxypropyl)methacrylamide (HPMA)ꢀbased polymers not exceeding 50 kDa, unless
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they are biodegradable . The size of such macromolecules is rather small to achieve an efficient
EPR effect and the synthesis of biodegradable HMW conjugates is not a simple task. HMW drug
carriers based on polymer micelles prepared by selfꢀassembly of smaller amphiphilic copolymers
(excretable in a form of unimer) offer a beneficial solution to this problem. Such nanoꢀsized
materials are usually prepared from block or graft copolymers consisting of hydrophilic and
hydrophobic chains of various lengths and compositions. Copolymers based on nonꢀreactive
poly(ethylene glycol)/poly(Lꢀlactide), poly(ethylene glycol)/poly(γꢀbenzylꢀLꢀglutamate) or
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poly(ethylene glycol)/poly(propylene glycol) are among the most examined ones
. These
copolymers have been described as carriers of hydrophobic drugs which, in most cases, were
nonꢀcovalently (hydrophobic interaction) incorporated into the hydrophobic core of the micelles
through the selfꢀassembly process. In spite of the structural and morphological variability of
these materials, the relatively difficult preparation of the defined micelles, together with their
tendency to aggregate, lower drug loading and complicated longꢀterm storage, limits their
clinical application.
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A suitable alternative to the above mentioned polymer micelles might be reversible
temperatureꢀsensitive micelles comprising a combination of hydrophilic and thermoꢀresponsive
block copolymers. The thermoꢀresponsive polymers are distinct in their ability to reversibly
change the chain morphology – from the random coil conformation (soluble form), to the
condensed globule (insoluble form) – depending on the temperature of the incubation media.
From the biomedical application point of view, the coilꢀglobule transition temperature (T ) of the
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thermoꢀresponsive polymer chain should be slightly below physiological temperature (37 °C) to
allow for the easy dissolution of the block copolymer in laboratory conditions and spontaneous
formation of the defined micelles immediately before (by heating up the solution to 37 °C) or
upon sample administration. Copolymers based on poly(Nꢀisopropylacrylamide)
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(poly(NIPAAm))
, poly(2ꢀisopropylꢀ2ꢀoxazoline) (poly(IPOX))
and elastin sideꢀchain
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polymers are examples of the most commonly studied materials meeting these requirements,
although the individual polymers exhibit particular pitfalls that might prevent their possible
application in the clinics. These limitations are the relatively strong dependence of the T on the
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degree of polymerization (poly(NIPAAm)), the concentration (elastinꢀbased polymers) and the
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relatively complex preparation technique that does not allow the synthesis of HMW uniform
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polymers (poly(IPOX))
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Considering the facts mentioned above, copolymers based on the poly[2ꢀ(2ꢀ
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methoxyethoxy)ethyl methacrylate] (poly(DEGMA)) seemed to be suitable candidates for the
proposed purposes as they fully meet the criteria for the biomedical application including the
nontoxicity, biocompatibility and transition temperature requirements (T ≈ 24 ꢀ 28 °C) and do
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not show the limitations observed in the case of the above mentioned materials
. In this
study, we used the unique combination of thermoꢀresponsive poly(DEGMA) with the highly
hydrophilic Nꢀ(2ꢀhydroxypropyl)methacrylamide copolymers (poly(HPMA)), which have often
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been applied as carriers for drug, protein or gene delivery
. The initial diꢀblock copolymer
precursors with various lengths of the thermoꢀresponsive poly(DEGMA) blocks were
synthesized by the reversible additionꢀfragmentation chainꢀtransfer (RAFT) polymerization
technique, enabling the preparation of the precisely defined materials. The poly(HPMA) block
contained ~5 mol. % of comonomer units with hydrazide groups intended for the attachment of
the cancerostatic drug pirarubicin (PIR). PIR was bound to the hydrophilic blocks of the
copolymers through the pHꢀsensitive hydrazone bond, which proved to be stable in the blood
stream at pH 7.4 but to undergo pHꢀsensitive hydrolysis in the intratumoral and intracellular
environment at pH below 6.5. The influence of the physicochemical parameters of the conjugates
on their in vitro biological behavior was investigated using the colorectal adenocarcinoma DLDꢀ
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cell lines. It was demonstrated that the studied conjugates effectively penetrated the cell
membranes of the cancer cells and released PIR inside the cells, which resulted in changed cell
morphology and damaged mutual intracellular connections, both indicating approaching cell
death.
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Experimental section
Materials
(RS)ꢀ1ꢀAminopropanꢀ2ꢀol, 6ꢀaminohexanoic acid (Ahx), 2,2′ꢀazobis(2ꢀmethylpropionitrile)
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(AIBN), 2ꢀcyanoꢀ2ꢀpropyl benzodithioate (CPB), 4ꢀcyanoꢀ4ꢀthiobenzoylsulfanylpentanoic acid
(CTP), N,N′ꢀdicyclohexylcarbodiimide (DCC), N,N‘ꢀdimethylacetamide (DMA), dimethyl
sulfoxide (DMSO), 1,4ꢀdioxan, methacryloyl chloride, 2ꢀ(2ꢀmethoxyethoxy)ethyl methacrylate
(DEGMA), tertꢀbutanol, tertꢀbutyl carbazate, triisopropylsilane (TIPS), trifluoroacetic acid
(TFA), 2,4,6ꢀtrinitrobenzenesulfonic acid solution (1 M in H2O) were purchased from Sigmaꢀ
Aldrich (SigmaꢀAldrich spol. s r.o., Czech Republic). Pirarubicin (PIR) was obtained from Meiji
Seika Pharma Co., Ltd. (Japan), and DYꢀ676 NHSꢀester was purchased from Dyomics GmbH
(Germany). All other chemicals and solvents were of analytical grade. Solvents were dried and
purified by conventional procedures and distilled before use.
The ELꢀ4 and DLD1 cell lines were obtained from ATCC (LGC Standards Sp. z o.o., Poland).
Dulbecco's Modified Eagle Medium (DMEM) and RPMIꢀ1640 media, Alamar Blue cell viability
reagent and Hoechst 33342 were purchased from Life Technology (Life Technologies Czech
Republic s.r.o., Czech Republic).
Sizeꢀexclusion chromatography (SEC)
The molecular weights and polydispersities of the polymers and polymerꢀPIR conjugates were
determined by SEC on a Shimadzu HPLC system equipped with UV–VIS diode array detector
(Shimadzu, Japan), refractive index OptilabꢀrEX, and multiangle light scattering DAWN EOS
detectors (Wyatt Technology Corp., Santa Barbara, CA). TSKꢀGel SuperAW3000 and
SuperAW4000 columns connected in series and 80 % methanol / 20 % sodium acetate buffer
(0.3 M, pH 6.5) as an eluent at a flow rate of 0.6 mL/min were used in all experiments. A method
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based on the known total injected mass with an assumption of 100 % recovery was used for the
calculation of the molecular weights from light scattering data. The numberꢀ and weightꢀaverage
molecular weights for the polymer precursors and the polymerꢀPIR conjugates are summarized
in Table 1 and Table 2, respectively.
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UV/VIS spectrophotometry
The spectrophotometric analyses were carried out in quartz glass cuvettes on a UV/VIS
spectrophotometer Helios Alpha (Thermospectronic, UK). The content of dithiobenzoate (DTB)
end groups in the polymers were determined at 302 nm in methanol using the molar absorption
DTB
coefficient ε
= 12,100 L/mol·cm. The content of hydrazide groups in the polymer precursors
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was determined using a modified TNBSA assay as described earlier. The results are
summarized in Table 1. The determination of the PIR content in the polymerꢀPIR conjugates
(without fluorophore) was performed at 488 nm in methanol using the molar absorption
PIR
coefficient ε = 11,300 L/mol·cm. The PIR contents are summarized in Table 2. The content of
carbonylthiazolidineꢀ2ꢀthione (TT) reactive groups in the polymer precursor was determined at
TT
305 nm using the molar absorption coefficient ε = 10,300 L/mol·cm.
Dynamic light scattering (DLS)
The hydrodynamic radii (RH) and scattering intensities (IS) of the polymer precursors and
polymer conjugates were measured by the DLS technique at a scattering angle θ = 173° using a
NanoꢀZS instrument (Model ZEN3600, Malvern Instruments, UK) equipped with a 632.8 nm
laser. The temperature measurements were performed to investigate the selfꢀassembly of
polymer coils to polymer micelles in the temperature range 20 ꢀ 50 °C (in 1 °C increments) in
PBS (1.0 mg/mL, pH 7.4) solutions. At each step, measurements were performed after reaching
the steady state conditions, which typically required approximately 10 min. For the evaluation of
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the dynamic light scattering data, the DTS(Nano) program was used. The mean of at least three
independent measurements was calculated. The transition temperature (T ) characterizing the
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polymer chain conformation changes was evaluated from the temperature dependence of the
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hydrodynamic diameter (D ); the T value was determined from the intersection point of two
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lines formed by the linear regression of a lower horizontal asymptote and a vertical section of the
Sꢀshaped curve (sigmoidal curve) fit. The T values for the polymer precursors and the polymerꢀ
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PIR conjugates are summarized in Table 1 and Table 2, respectively.
PIR release assays
The release of PIR from the polymerꢀPIR conjugates was measured in aqueous solutions at 37
°
C at two different pH values using a monochromatorꢀbased multiꢀmode microplate reader
Synergy H1 (BioTek, USA). Specifically, the polymer conjugates (c = 2.5 mg/mL) were
dissolved in 0.15 M PBS (pH 5.5 or 7.4), heated up to 37 °C and kept at this temperature for the
duration of the analysis. In the selected time intervals, the aliquots (140 ꢁL) were loaded on the
preheated PD SpinTrap Gꢀ25 columns (GE Healthcare, UK) and centrifuged at 37 °C for 2
minutes at 800 × g. Subsequently, the absorbances of the collected polymer fractions were
measured in 96ꢀwell plates at 486 nm. The PIR release from the polymerꢀPIR conjugates was
plotted as the relative decrease of amount of polymerꢀbound pirarubicin over time. An
absorbance of the appropriate polymerꢀPIR conjugate measured immediately upon its dissolution
and subsequent centrifugation was used as an initial value (A(t0)).
The stabilities of the polymerꢀPIR conjugates in the buffers modeling both extracellular and
intracellular environments were also evaluated using a SEC column on the Shimadzu HPLC
system (see above). Specifically, the polymer conjugates (c = 1.0 mg/mL) were dissolved in 0.15
M PBS (pH 5.5 or 7.4), heated up to 37 °C and kept at this temperature for the duration of the
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analysis. At selected time intervals, the aliquots (20 ꢁL) were loaded on the TSKꢀGel
SuperAW3000/4000 column and eluted using the 80 % methanol / 20 % sodium acetate buffer
(0.3 M, pH 6.5) mixture. The relative amount of the polymerꢀbound PIR was evaluated from the
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polymer peak area recorded by the UV–VIS detector at 486 nm. The release of PIR from the
conjugates was plotted as a change of the polymerꢀbound PIR concentration over time.
Isothermal Titration Calorimetry (ITC)
The critical micellar concentration (CMC) values were determined by an isothermal titration
microcalorimeter MicroCal iTC200. The ITC experiments were performed using either 20
injections of the polymer solution in PBS buffer above the T into PBS buffer (a constant
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titration volume of 2 µL; 180 s intervals). The thermograms were recorded and analyzed using
Origin 7 software.
Cell line cultures
ELꢀ4 murine Tꢀcell lymphoma cell line was cultured in DMEM supplemented with heat
inactivated 10 % fetal calf serum (FCS), penicillin (100 U/mL) and streptomycin (100 ꢁg/mL).
The DLDꢀ1 human colorectal adenocarcinoma cell line was cultured in RPMIꢀ1640 medium
with heat inactivated 10 % FCS, penicillin (100 ꢁg/mL) and streptomycin (100 ꢁg/mL).
In vitro cell viability assay
DLD1 (5 × 104) or EL4 cells (15 × 104) were seeded in 100 µL media into 96ꢀwell flatꢀbottom
plates 24 h before the addition of the polymerꢀPIR conjugates or free PIR. The polymerꢀPIR
conjugate stock solutions (5 mg/mL in PBS) were diluted with PBS to concentrations ranging
from 0.2 to 200 µg/mL; PIR was firstly dissolved in DMSO (10 mg/mL) and then diluted with
PBS to 0.01 – 100 µg/mL. Thereafter, 10 µL of the polymerꢀPIR conjugates or PIR were added
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to the cells and the cells were cultured for 72 h in 5 % CO atmosphere at 37°C. Then, 10 µL of
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to the highly fluorescent compound resorufin. The fluorescence of resorufin was detected using
the Synergy Neo plate reader (BioTek, USA) at 570/610 nm (excitation/emission). The cells
cultured in a medium without Alamar Blue were used as negative controls. Three wells were
used for each concentration. The assay was repeated three times independently. Statistical
analysis for oneꢀway ANOVA was performed using the GraphPad Prism Software. The IC50
values for the polymerꢀPIR conjugates and free PIR are summarized in Table 3.
Internalization of fluorescently labeled polymerꢀPIR conjugates
DLD1 cells were cultured for 24 h in 5 % CO atmosphere at 37°C on a 35ꢀmm glass bottom
2
dish with 4 chambers, a 20 mm microwell, and a #1 cover glass (0.13ꢀ0.16 mm). The amount of
the fluorescently labeled polymerꢀPIR conjugate added to the cell suspensions was normalized to
the DYꢀ676 content (1 ꢁg DYꢀ676/mL), corresponding to the final conjugate concentration of
100 ꢁg/mL. After 2, 4, 8, 16, 24 and 48 h, the native cells were washed three times with PBS and
the nuclei were with 5 ꢁg/mL of Hoechst 33342. DYꢀ676ꢀlabeled polymer conjugates were
excited at 674 nm and the emitted light was detected at 699 nm. PIR was excited at 488 nm and
the emission was measured through a 500 – 600 nm filter. Hoechst 33342 dye was excited at 405
nm and the emitted light was measured through a 450 – 500 nm filter. The fluorescence and
transmitted light was acquired using a laser scanning confocal microscope (LSCM) Olympus
IX83 with the FV10ꢀASW software (Olympus, Czech Republic). The samples were scanned with
a 60 × oil immersion objective Plan ApoN (1.42 numerical aperture; Olympus, Czech Republic).
Competitive Hoechst staining ꢀ detection of doxorubicin in cell nuclei
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DLDꢀ1 cells were incubated with free PIR or with the polymerꢀPIR conjugates at
concentration 100 – 0.01 ꢁg/mL of PIR under standard culture conditions. After 24 h, the cells
were detached with a detaching solution (100 mM HEPES, 20 mM NaCl, 10 mM EDTA, 0,5 %
BSA), stained with Hoechst 33342 (10 ꢁg/mL) at 37 °C for 30 min and washed with iceꢀcold
PBS with BSA (0.5 %). The cells were immediately analyzed using a BD FacsVerse flow
cytometer (BD Biosciences, USA). The excitation wavelength for PIR was 488 nm and the
emission was measured through a 586BP42 pass filter; the Hoechst 33342 was excited at 405 nm
and the emission was detected through a 448BP45 filter. The data were analyzed in two
independent experiments. The medians of the fluorescence intensities were calculated using
FlowJo software (Tree Star, USA).
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Synthesis of monomers
Nꢀ(2ꢀHydroxypropyl)methacrylamide (HPMA) was synthesized by reacting methacryloyl
chloride with (RS)ꢀ1ꢀaminopropanꢀ2ꢀol in dichloromethane in the presence of sodium carbonate
2
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as described .
Nꢀ(tertꢀbutoxycarbonyl)ꢀN’ꢀ(6ꢀmethacrylamidohexanoyl)hydrazine (MaꢀAhxꢀNHNHꢀBoc)
was prepared in twoꢀstep process by the reaction of methacryloyl chloride with 6ꢀaminohexanoic
acid in the presence of NaOH followed by the condensation of formed 6ꢀ
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(
methacryloylamino)hexanoic acid with tertꢀbutyl carbazate in the presence of DCC .
NꢀMethacryloylglycylphenylalanylleucylglycine (MAꢀGFLGꢀOH) was prepared by automated
solid phase peptide synthesis on 2ꢀchlorotrityl chloride resin starting from the Cꢀterminus using
standard Fmoc procedures. 3ꢀ(NꢀMethacryloylglycylphenylalanylleucylglycine)thiazolidineꢀ2ꢀ
thione (MaꢀGFLGꢀTT) was prepared by reacting MaꢀGFLGꢀOH with 4,5ꢀdihydrothiazoleꢀ2ꢀthiol
30
in DMF in the presence of DCC according to .
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Synthesis of diꢀblock copolymer precursors PP2 – PP4
The thermoꢀresponsive micelleꢀforming polymer precursors were produced as AꢀB type diꢀ
block copolymers by RAFT polymerization in two synthetic steps. The hydrophilic block A was
prepared by copolymerizing HPMA with MaꢀAhxꢀNHNHꢀBoc using CPB as a chain transfer
agent and AIBN as an initiator. The hydrophilic block A was subsequently subjected to a chainꢀ
extension polymerization through the RAFT mechanism with DEGMA to introduce the thermoꢀ
responsive polymer block B. Three different ratios of poly[(HPMA)ꢀcoꢀ(MaꢀAhxꢀNHNHꢀBoc)],
as the macroꢀchain transfer agent, to DEGMA were used to synthesize the diꢀblock polymers
with variable lengths of the thermoꢀresponsive blocks.
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Example: A mixture of 5.8 mg CPB (26.1 ꢁmol) and 2.1 mg AIBN (13.0 ꢁmol) was dissolved
in 0.368 mL of DMSO and added to a solution of 500.0 mg HPMA (3.49 mmol) and 57.6 mg
MaꢀAhxꢀNHNHꢀBoc (0.18 mmol) in 3.312 mL of tertꢀbutanol. The reaction mixture was
thoroughly bubbled with Ar and polymerized in the sealed glass ampoules at 70 °C for 6 h. The
resulting copolymer was isolated by precipitation into a 3:1 mixture of acetone and diethyl ether
TM
and purified by gel filtration using a Sephadex LHꢀ20 cartridge in methanol. The methanolic
solution was concentrated under reduced pressure and precipitated in diethyl ether yielding 268.3
mg (48.1 %) of the poly[(HPMA)ꢀcoꢀ(MaꢀAhxꢀNHNHꢀBoc)] polymer precursor (PP1) as a pink
powder. The content of dithiobenzoate (DTB) end groups was nDTB = 60.3 ꢁmol/g,
corresponding to Mn,UV=16,580 g/mol (f = 0.85). Next, a solution of 50.0 mg PP1 (3.01 ꢁmol
DTB groups) in 64.8 ꢁL of deionized H O was added to a mixture of 21.6 mg DEGMA (0.12
2
mmol) and 0.1 mg AIBN (0.60 ꢁmol) in 130 ꢁL of 1,4ꢀdioxane, thoroughly bubbled with Ar and
allowed to polymerize in the sealed glass ampoules at 70 °C for 18 h. The diꢀblock polymer was
isolated by precipitation to diethyl ether yielding 63.8 mg (89.1 %) of the pale pink amorphous
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solid. The precipitated polymer was dissolved in 638 ꢁL of DMSO, 16.0 mg of ABIN (97.4
ꢁ
mol) was added and the solution was heated up to 80 °C for 2 more hours. The resulting diꢀ
block copolymer was isolated by precipitation in diethyl ether and purified by gel filtration using
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the Sephadex LHꢀ20 cartridge in methanol. The methanolic solution was concentrated under
vacuum and precipitated in diethyl ether yielding 52.1 mg of the poly[(HPMA)ꢀcoꢀ(MaꢀAhxꢀ
NHNHꢀBoc)]ꢀblockꢀp(DEGMA) polymer as a white amorphous solid. The diꢀblock copolymer
with Bocꢀprotected hydrazide groups was suspended in 700 ꢁL of TFA/TIPS/ H2O (92 : 2.5 :
2.5) mixture and sonicated until all solids were dissolved (~5 min). Thereafter, the polymer was
precipitated into diethyl ether and reꢀdissolved in phosphate buffer (0.15 M, pH 8.0). The pH of
the solution was adjusted to 7.4 using a NaOH solution and the polymer was desalted by the
TM
Sephadex PDꢀ10 column in H O. The resulting polymer precursor poly[(HPMA)ꢀcoꢀ(Maꢀ
2
AhxꢀNHNH )]ꢀblockꢀp(DEGMA) (PP2) was isolated from an aqueous solution by lyophilization
2
yielding 48.3 mg of the white solid. The reaction conditions for all synthesized polymer
precursors (PP1 – PP4) as well as their physicochemical characteristics are summarized in the
Table 1.
Synthesis of hydrophilic copolymer precursor PP6
The copolymer precursor poly[(HPMA)ꢀcoꢀ(MaꢀGFLGꢀTT)] (PP6) was prepared by RAFT
polymerization of 200.0 mg HPMA (1.40 mmol) and 87.0 mg MaꢀGFLGꢀTT (0.15 mmol) using
0.64 mg AIBN (3.90 ꢁmol) as an initiator and 1.72 mg CTP (6.16 ꢁmol) as a chain transfer
agent. The polymerization mixture was dissolved in 1.563 mL of tertꢀbutanol with 10 % DMSO,
transferred into a glass ampoule, bubbled with Ar and sealed. After 24 h at 70 °C, the product
was isolated by precipitation of the reaction mixture to acetone/diethyl ether (3:1); the precipitate
was then washed with diethyl ether and dried under vacuum. This product was then reacted with
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AIBN (10 molar excess) in DMSO (15% w/w solution of polymer) under Ar for 3 h at 70 °C in a
sealed ampoule to remove DTB end groups. The reaction mixture was isolated by precipitation
in acetone/diethyl ether (3:1); the precipitate was washed with diethyl ether and dried under
vacuum to yield 186.5 mg of copolymer precursor PP6. The numberꢀaverage molecular weight
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(M ) of the copolymer was 35,500 g/mol and polydispersity index (Ð) was 1.12. The content of
n
TT
TT groups (φ ) was 6.8 mol. %.
Attachment of PIR and fluorophore to the diꢀblock copolymer precursors (PC2F – PC4F)
Pirarubicin was attached to the hydrazide groups distributed along the hydrophilic block A of
the copolymer precursors through the hydrolytically degradable hydrazone bond. For the FACS
analysis (see above), some of the hydrazide groups of the copolymers were used for the
copolymer labeling with a fluorescent dye via the stable diacylhydrazine bonds.
Example: A mixture of 20.0 mg PP2 and 0.4 mg DYꢀ676 NHSꢀester (0.44 ꢁmol) was
dissolved in 80 ꢁL of DMA and the solution was stirred for 4 h at r.t. Then, 2.2 mg PIR (3.5
ꢁ
mol) in 80 ꢁL of methanol and 8 ꢁL of acetic acid was added dropꢀwise and the reaction
mixture was stirred for another 18 h at r.t. The resulting polymer conjugate (PC2-F) was
TM
separated from the reaction mixture by gel filtration using the Sephadex LHꢀ20 column in
methanol followed by inspissation of the methanolic solution on a rotary evaporator and
precipitation of the conjugate in ethyl acetate yielding 12.6 mg of the dark violet solid. The
physicochemical characteristics for the polymerꢀPIR conjugates are summarized in Table 2.
Attachment of PIR to the hydrophilic copolymer precursor (PC6)
A mixture of 92.0 mg PP6 (36.5 ꢁmol TT) and 7.9 mg PIR (12.6 ꢁmol) was dissolved in 1.75
mL of DMA and stirred at r.t. The yield of the reaction was monitored by HPLC. After 3 h, when
the reaction was completed, 4.3 ꢁL of (RS)ꢀ1ꢀaminopropanꢀ2ꢀol (54.8 ꢁmol) was added to the
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reaction mixture to quench the unreacted TT. After another 30 min, the polymer conjugate was
isolated by precipitation of the reaction mixture in ethyl acetate and the precipitate was
centrifuged. The precipitate was washed with diethyl ether and dried under vacuum yielding 84.0
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PIR
mg of PC6. M = 41,000 g/mol, Ð = 1.17 and ω = 9.0 wt. %. The chemical structure of PC6
n
conjugate si shown in Figure S1 B.
Results and discussion
PolymerꢀPIR conjugates synthesis
The synthesis of the polymerꢀPIR conjugates was carried out in two synthetic steps, including
the synthesis of the amphiphilic diꢀblock copolymer precursors, followed by the attachment of
either the cancerostatic drug pirarubicin or the fluorescent dye. The precursors (PP1 – PP4) were
synthesized by the RAFT polymerization technique, enabling the synthesis of highly defined
uniform materials with predetermined molecular weights and high yield of DTB endꢀgroup
content. The hydrophilic block (PP1) was prepared by copolymerizing HPMA with MaꢀAhxꢀ
NHNHꢀBoc using the DTBꢀderived chain transfer agent (see Scheme 1). The copolymer with Mn
=
14,100 g/mol was distinguished by narrow molecular weight distribution (M /M = 1.11) and
w n
sufficient functionality (f = 0.85) of the DTB end groups. The functionality of the polymer (f, the
amount of the functional end groups per one polymer chain) was defined as the ratio between Mn
obtained from the SEC measurement and Mn,UV calculated from the end group analysis. The
content of comonomer units bearing the hydrazide groups, determined by TNBSA assay (after
1
removal of Boc protective groups, see Experimental section) and confirmed by HꢀNMR
spectroscopy (data not shown) was 4.8 mol. %, which is equal (within experimental error) to the
quantity of MaꢀAhxꢀNHNHꢀBoc (5.0 mol. %) in the polymerization feed. The precursor PP1
was further used as the macroꢀchain transfer agent for the subsequent chain extension with the
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thermoꢀresponsive poly(DEGMA) blocks. Three different ratios of DEGMA to DTBꢀend groups
of PP1 were used (see Table 1) to synthesize the amphiphilic diꢀblock copolymers with variable
lengths of the thermoꢀresponsive block ranging from approximately 7,000 to 24,600 g/mol. The
incorporation of poly(DEGMA) blocks to the HPMAꢀbased copolymer precursor resulted in a
moderate broadening of the distribution of molecular weights (M /M ≈ 1.5). This can be
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ascribed to a low but not insignificant quantity of PP1 dead chains (not terminated with DTB
groups) that could not react further with DEGMA through the RAFT mechanism. Consequently,
the resulting precursors were the mixture of two polymer populations with different molecular
weights – the largely prevailing diꢀblock copolymer and a small amount of initial hydrophilic
precursor (Figure 1).
To prevent the unwanted reactions of DTB end groups of the polymers with the hydrazide side
chain groups during the removal of Boc protective groups or with PIR during the following
conjugation, the DTB groups were removed by a reaction with a high molar excess of AIBN
through the homolytic mechanism. The replacement of DTB groups by the isobutyronitrile
groups was documented by the disappearance of the UV absorption peak at 302 nm. Finally, Boc
groups protecting the hydrazides were quantitatively removed using the TFA/TIPS/H2O mixture,
commonly used in peptide chemistry, yielding the polymer precursors PP2 – PP4. Neither the
DTB group substitution nor the removal of Boc groups caused significant changes in the
molecular weights and molecular weight distributions of the polymer precursors. The molecular
weight parameters for PP2 – PP4 are summarized in Table 1 and SEC profiles are depicit in
Figure 1.
The conjugation of PIR to the copolymer precursors was achieved by the hydrazone bonds
formed between the keto group of PIR and the hydrazide groups in the side chains of polymers
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(
the reaction scheme is shown in Scheme 2, an example of SEC chromatogram for the conjugate
PC2 recorded by LS, RI and UV detectors is depicit in Figure S2 B). Based on our previous
experiences with the structurally similar cancerostatic drug doxorubicin (DOX), the reaction was
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performed in methanol in the presence of acetic acid . The yields of the conjugation reaction
ranged between 70 and 80 %. The method of PIR attachment was selected with respect to the
pHꢀcontrolled stability of the hydrazone bond – relatively stable at neutral pH but susceptible to
much faster hydrolysis in mildly acidic conditions. This approach is very beneficial for a
systemic administration because the majority of the drug remains attached to the polymer carrier
during its transport in the bloodstream (pH 7.4) but is released in the target tumor cells (pH 5.5).
To verify this claim, we measured the release of PIR from conjugates PC2 – PC4 under
physiological environmentꢀmodeling conditions (see Experimental section) at both relevant pH
values. As shown in Figure 2A, the rate of the hydrazone bond hydrolysis proceeded much faster
in solutions with a lower pH, which documents the suitability of the conjugates for safe drug
delivery purposes. Although the ability of the conjugates to release PIR under the conditions
modeling the intratumoral environment is evident, it appears that the conjugates in aqueous
solution still contain approximately 35 – 40 % of bound PIR even after 24 h of acidic hydrolysis
at pH 5.5. This can be explained by the hydrophobic character of the poly(DEGMA) chains
retaining the already released hydrophobic drug packed inside the polymer coil by hydrophobic
interactions upon disruption of the hydrazone bond . The hydrophobic retention of PIR by the
polymer coil increased with increasing poly(DEGMA) block length. The release of PIR was also
evaluated by GPC analysis with the methanol/sodium acetate buffer (80:20) mixture as a mobile
phase (see Experimental section). In the methanolic solution, the hydrophobic interaction
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between the polymer and the released drug was disrupted, which resulted in an almost complete
release of PIR from the carrier by hydrolysis (see Figure 2B).
For the laser scanning confocal microscopy (LSCM) measurement of the interaction of the
conjugates with the cells, the polymer precursors PP2 – PP4 were modified (in a oneꢀpot
reaction together with PIR) with DYꢀ676 NHS ester fluorescent dye (PC2F – PC4F) that emits
light in a different region of the light spectrum than PIR (see Experimental section). Because the
fluorescent dye was attached to the polymer backbone of the conjugates through the stable
diacylhydrazine bonds (see Scheme 2), the LSCM images enabled a simultaneous observation of
the intracellular fate of the polymer and PIR (both bound and released) over time. However, we
would like to note that the apparent molecular weights of the fluorescently labeled polymerꢀPIR
conjugates, measured by GPC equipped with a LS detector, were approximately 1.5 times higher
than those of the corresponding polymer precursors. Because no broadening of the molecular
weight distributions was observed, we hypothesize that this difference was due to the
contribution of PIR fluorescence to the intensity of the light scattering. Therefore, it was
necessary to calculate the molecular weights by a "relative" molecular weight determination
method using a series of wellꢀcharacterized poly(HPMA) polymer standards. The molecular
weights of such characterized conjugates were only approximately 3 ꢀ 5 % higher than those of
the corresponding precursors, which is in good agreement with the theoretical expectation.
Complete physicochemical characteristics of the polymerꢀPIR conjugates, including the
corrected MWs are summarized in Error! Reference source not found..
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Solution behavior of thermoꢀresponsive polymerꢀPIR conjugates
The temperatureꢀdependent changes in solution behavior of the polymerꢀPIR conjugates and
their corresponding precursors were studied by DLS. We focused on the influence of the
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composition and structural parameters of the polymers on their hydrodynamic properties in the
solutions mimicking physiological conditions as our understanding of such properties may play
an important role in the design of drug delivery systems for efficient in vivo treatment. First, we
clearly demonstrated that the hydrophobic poly(DEGMA) block length (MW) is the decisive
structural factor. Specifically, an increase in molecular weight of the poly(DEGMA) block (at a
constant MW of the hydrophilic block) caused not only a decrease in transition temperatures (Ttr)
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of the copolymers but also resulted in the formation of largerꢀsized polymer coils (below T ) and
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largerꢀsized polymer micelles (above T ) (see Figure 3). For example, the PP2 precursor with
tr
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8 °C higher than poly(DEGMA) homopolymer (T ≈ 26 °C), while in the case of PP4, where
tr
the MW of the hydrophobic block is 1.7 times higher than MW of the hydrophilic block, the Ttr
of the copolymer is only slightly higher in comparison to poly(DEGMA) itself. Based on these
observations (and also our understanding of the dependence of T on MW of other thermoꢀ
tr
responsive polymers) it is reasonable to predict that further poly(DEGMA) chain lengthening
would not result in significant T decrease. Additionally, an increase in the polymer micelle sizes
tr
of the PC2 – PC4 conjugates with increasing MW of poly(DEGMA) chains was predictable
because longer polymer chains selfꢀassembled into largerꢀsized nanoparticles. The length of the
hydrophobic block has also an impact on the micelle stabilities expressed by the critical micellar
concentration (CMC) values. It was shown that the CMC values decreased (stabilities of the
conjugates increased) with increasing MW of the poly(DEGMA) blocks from 0.071 mg/mL for
PC2 up to 0.022 mg/mL for PC4. Considering the usual concentrations (~10 mg PIR/kg of
mouse body weight, i.v. injection) of the previously described polymerꢀPIR conjugates in mice
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experiments , the CMC values acquired for PC2 – PC4 guarantee, with a sufficient reserve,
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that the thermoꢀresponsive conjugates will exist in their micellar form even after their injection
to the mice. This interesting finding brought a comparison of T of the polymer precursors and
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the polymerꢀPIR conjugates. As shown in Figure 3, the incorporation of the hydrophobic drug to
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the polymers caused a marked decrease in T (by ~5 °C) regardless of the poly(DEGMA) block
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length. Because PIR is attached to the polymers through hydrolytically degradable bonds, it is
reasonable to expect that T of the conjugates will increase upon PIR release inside the cells (an
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example of increase of T of the conjugate PC2 after 24 h of hydrolysis carried out under model
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conditions (PBS, pH 5.5, 37 °C) is shown in Figure S3). This increase in T can be employed in
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the design of biodegradable micelleꢀbased drug delivery systems. Such drugꢀbearing conjugates
can reach the target tumor cells in their micellar form (T < 37 °C) through the EPR effect, but
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the drugꢀfree copolymer (T > 37 °C) can be reliably eliminated from the body as an unimer
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(single copolymer molecules) formed upon PIR release, through renal filtration. From this point
of view, the conjugate PC3 exhibited the most favorable properties. It formed a polymer micelle
spontaneously under physiological conditions, but began to change its morphology to random
coil during PIR cleavage from the polymer. As shown at Figure 4, polymer conjugate PP3 (the
precursor of PC3) at 37 °C is predominantly in the micellar form, however, a significant portion
of the polymer is present already in the form of unimers. To the contrary, the conjugate PC2
adopted the random coil formation both before and after PIR release and the conjugate PC4
existed only as a fully solvated unimer regardless of attached or released PIR (see Figure 3 and
4).
Cytotoxicities of the polymerꢀPIR conjugates
The cytotoxicities of PIR and the polymerꢀPIR conjugates were reported as IC values, which
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is the concentration of material at which 50 % of the cell culture is no longer viable. In this
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experiment, we used the human colorectal adenocarcinoma (DLDꢀ1) and the mouse lymphoma
(
ELꢀ4) cell lines, both relevant models to this study. According to the results in Table 3, the IC50
values of the thermoꢀresponsive polymerꢀPIR conjugates PC2 – PC4 are all very similar
approximately 3 ꢁg/mL against DLDꢀ1 cell line and 0.01 ꢁg/mL against ELꢀ4 cell line).
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However, these values are approximately 20 times higher (in the case of DLDꢀ1 cells) than those
for free PIR. This finding demonstrates that the attachment of the drug to the polymer carriers
significantly reduces its direct cytotoxicity and, at the same time, that the poly(DEGMA) block
length nowise affects the toxicity of the conjugates. Lower conjugate toxicity is favorable
because it enables the administration of the drug at a higher concentration, thus increasing the
potential therapeutic effect. The comparison of the thermoꢀresponsive polymerꢀPIR conjugates
PC2 – PC4 with the linear hydrophilic polymerꢀPIR conjugates containing the drug attached to
the polymer backbone either through the hydrolytically degradable hydrazone bond (PC5) or
through the enzymatically degradable tetrapeptide spacer GFLG (PC6) led to interesting results.
While the cytotoxicity of the conjugate PC5 was approximately 2 times (for the DLDꢀ1 cells), or
100 times (for the ELꢀ4 cells) higher than that for PC2 – PC4 (not statistically significant
differences), the conjugate PC6 was approximately 5 times less toxic (for DLDꢀ1 cells) than the
thermoꢀresponsive ones (statistically significant differences). The higher toxicity of the PC5
conjugate can be ascribed to the more hydrophilic character of its polymer backbone enabling
faster release of PIR from the carrier without its subsequent retention due to hydrophobic
33
interaction with the hydrophobic block of the copolymer (see ref. ). To the contrary, conjugate
PC6, although having PIR bound via the enzymatically degradable GFLG linker, released only a
modest amount of the drug within the monitored time interval. This is in agreement with a
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published observation
. The polymer precursors PP2 – PP4 showed no toxicity. The
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pronounced differences of approximately two orders of magnitude in the IC values measured in
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DLDꢀ1 and ELꢀ4 cells are caused by the different sensitivity of the two cell lines to PIR.
Internalization of the polymerꢀPIR conjugates into cells
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The intracellular fate of the polymerꢀPIR conjugates and free PIR was observed in DLDꢀ1
cells by LSCM at different time points (see Experimental Section). The fluorescent signal of the
DYꢀ676ꢀlabeled polymer conjugates increased over time for all studied samples. As shown in
Figure 5, there are obvious differences in the intracellular localization and the mechanism of
action between free PIR, the thermoꢀresponsive polymerꢀPIR conjugates and the control
hydrophilic polymerꢀPIR conjugates. While in the case of free PIR and the control polymerꢀPIR
conjugate having PIR attached through the hydrolytically degradable bond (PC5) a substantial
amount of the drug was detected in the cytoplasm and inside the cell nuclei, in the case of the
thermoꢀresponsive polymerꢀPIR conjugates (PC2 – PC4) and the control hydrophilic polymerꢀ
PIR conjugate having PIR attached through the enzymatically degradable GFLG spacer (PC6)
PIR was predominantly detected in the cytoplasm and on the surface of the nuclear membrane.
The fact that only a very small amount of PIR was found in the cell nuclei can be probably
attributed to the hydrophobic retention of the released PIR in the hydrophobic poly(DEGMA)
domains of copolymers PC2 – PC4 and the relatively slow enzymatic cleavage of the GFLG
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tetrapeptide spacer in copolymer PC6 by lysosomal proteases
. Surprisingly, although the
structure and chemical composition of the two types of conjugates (PC2 – PC4 and PC6) is
different, their intracellular fate, according to the LSCM study, seems to be quite similar. These
polymer/PIR conjugates probably interact hydrophobically with the lipid bilayer of the cell
nucleus, thereby decreasing the membrane permeability and preventing further progression of the
cells. LSCM showed that the cells incubated with the PC2 – PC4 and PC6 conjugates
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significantly changed their morphology and size of the intracellular compartments, namely the
nuclei, in comparison with the nonꢀtreated cells (not shown) and the cells treated with PIR or
PC5 conjugate. This behavior of the cells resembles the phenomenon called oncosis, a nonꢀ
apoptotic mode of cell death, characterized by mitochondrial swelling, cytoplasm vacuolization,
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and swelling of the nucleus and cytoplasm . Moreover, the cells treated with PC2 – PC4 and
PC6 conjugates showed reduced amount of the mutual intracellular connections documenting an
apparent damage of the cells. Because the polymer precursors PP2 – PP4 without PIR were nonꢀ
toxic and did not affect the morphology of the cancer cells (LSCM images not shown), it is
obvious that the polymer carriers alone were not responsible for the observed cytotoxic effect of
the conjugates. In this experimental setting, no pronounced difference in the behavior of cells
treated with individual thermoꢀresponsive polymerꢀPIR conjugates PC2 – PC4 and polymer
PC6 on the cancer cells was observed. It appears that the poly(DEGMA) block, regardless of its
length, is the principal structural factor of the polymerꢀPIR conjugates influencing their
biological behavior. As already mentioned, despite the significant structural differences between
the thermoꢀresponsive polymerꢀPIR conjugates PC2 – PC4 and hydrophilic polymerꢀPIR
conjugate PC6, the LSCM images documenting the intracellular fate of the conjugates are
strikingly similar. We can hypothesize that both types of conjugates exhibit an amphiphilic
character (due to the presence of thermoꢀresponsive poly(DEGMA) block in conjugates PC2 –
PC4 and a hydrophobic GFLG spacer between PIR and polymer backbone in conjugate PC6)
that might be responsible for the nonꢀspecific interaction of the polymer conjugates with various
membrane structures inside the cells. The detailed analysis of the intracellular behavior of the
conjugates and the mechanism of their action in the cancer cells will be the subject of our
upcoming publication.
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Incorporation of PIR into the cell nuclei
To verify the results from LSCM images, the Hoechst incorporation assay using FACS
analysis was performed. The cells (DLDꢀ1) were incubated with a concentration array of the
polymerꢀPIR conjugates or free PIR for 24 h followed by the addition of Hoechst 33342, an
intercalating fluorescent dye used for DNA staining. The assay is based on the characteristics of
PIR to intercalate between the base pairs of DNA strands and thus block the subsequent
interaction of DNA with Hoechst. In other words, the Hoechst dye can insert only in sections of
DNA strands that are not occupied by PIR. The results of intercalating competitions between the
DNA/PIR complexes and the Hoechst 333258 dye for the individual polymerꢀPIR conjugates as
well as for free PIR are shown in Figure 6. The highest intercalation ability showed free PIR
whose concentration in the cell nuclei gradually increased (the concentration of Hoechst in the
cell nuclei gradually decreased) from 1 ꢁg/mL to 100 ꢁg/mL, at which point almost all
intercalating sites of DNA were occupied by PIR (no Hoechst was detected). A slightly lower
intercalation efficiency was found in the case of PC5 conjugate where a sharp increase in PIR
fluorescence intensity was observed starting from 10 ꢁg/mL of PIR. However, as in the previous
case, DNA was fully saturated with PIR at a concentration of 100 ꢁg/mL. This demonstrates the
successful hydrolysis of the hydrazone bond, release of PIR from the conjugate and its
subsequent penetration in the nucleus. Markedly different results were obtained when the cells
were incubated with the PC6 conjugate. Although PIR started incorporating slightly in the nuclei
at a concentration 1 ꢁg/mL, the efficiency of its intercalation was relatively low, which enabled
the Hoechst dye to interact with DNA too. Only in the case of the highest concentration of the
conjugate (100 ꢁg/mL of PIR), the amount of incorporated Hoechst decreased. This can be
ascribed to a very low rate of PIR release from the conjugate due to the relatively slow gradual
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enzymatic cleavage of the GFLG spacer between the drug and the polymer backbone
and
possibly also the hydrophobic retention of the released drug within the polymer coil containing
amphiphilic GFLG peptides. Similar results were also observed in cells incubated with the
thermoꢀresponsive PC2 – PC4 conjugates. In all cases, the Hoechst dye was incorporated in
DNA in the entire range of concentration of the conjugates (0.01 – 100 ꢁg/mL of PIR),
indicating the low level of PIR intercalated in the cell nuclei even at the highest conjugate
concentration. These results are in good agreement with the LSCM pictures, in which almost no
PIR signal from the PC2 – PC4 conjugates was detected inside the nuclei. This confirms our
hypothesis that PIR remains hydrophobically entrapped inside the coil of the thermoꢀresponsive
polymers even after the hydrazone bond hydrolysis inside the cells. Furthermore, it seems that
this interaction is independent of the poly(DEGMA) block length, or more precisely, of the
conjugate morphology.
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Conclusion
Conjugates of nanoꢀsized polymer carriers with chemotherapeutics are considered to be a very
promising drug delivery system intended for the treatment of neoplastic diseases. The major
benefit is in their high ability for passive accumulation in solid tumors due to the EPR effect,
while causing minimal damage to healthy tissues. However, the clinical application of the
majority of the nanoparticleꢀbased carriers encounters obstacles related to their relatively
laborious preparation, high tendency to aggregate, limited drug loading and complicated longꢀ
term storage. Therefore, in this work, we focused on the development of novel thermoꢀ
responsive polymer carriers characterized by relatively easy and reproducible preparation,
precisely defined chain structure with multiple binding sites for covalent attachment of drugs, an
adjustable unimer ꢀ micelle transition temperature and longꢀterm stability. The diꢀblock polymer
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carriers, synthesized by RAFT polymerization, were comprised of a multivalent HPMAꢀbased
hydrophilic block and a thermoꢀresponsive DEGMAꢀbased block of various lengths. The
cancerostatic drug pirarubicin (PIR) was conjugated to the hydrophilic blocks of the copolymers
via a hydrolytically cleavable hydrazone bond. The polymerꢀPIR conjugates showed
spontaneous pHꢀdriven hydrolysis of the hydrazone bonds; PIR release proceeded much faster at
pH 5.5, corresponding to the intracellular environment than at pH 7.4, corresponding to the blood
circulation, thus meeting the requirements for safe delivery of the drug to cancer cells. The
conjugates underwent reversible temperatureꢀinduced conformation changes from random coils
to stable nanoꢀsized micelles; the micelle size and stability as well as the transition temperature
of the conjugates were driven by the thermoꢀresponsive block length and PIR content in the
range of 26 to 39 °C. All studied conjugates, regardless of thermoꢀresponsive block length,
showed significantly lower cytotoxicity than free PIR (both in DLDꢀ1 and ELꢀ4 cells) and
demonstrated the ability to effectively penetrate the cell membrane of model cancer cell lines
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(DLDꢀ1). Although a considerable amount of PIR remained hydrophobically attached to the
polymer carrier even after the hydrazone bond disruption inside the cells, the resulting
polymer/PIR associates surrounded the cell nuclei membranes and blocked the transport through
the membrane. This led to changes in cell morphology and disrupted mutual intracellular
connections, indicating approaching cell death. Based on these encouraging results, we believe
that the studied conjugates have a great potential to become efficacious in vivo pharmaceuticals.
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Figure 1. SEC profiles of the copolymer precursors PP1 – PP4 plotted as a function of time of
the normalized RI responses.
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Figure 2. Release of PIR from the polymerꢀPIR conjugates PC2 – PC4 incubated in phosphate
buffers at pH 7.4 (dashed lines) and pH 5.5 (solid lines) at 37 °C. Quantification of the polymerꢀ
bound PIR was performed using the UV/VIS spectrophotometry by measuring the absorbance of
the polymerꢀPIR conjugates separated from free PIR by GPC carried out either in (A) PBS or in
(B) methanol/sodium acetate buffer (80:20) mixture.
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Figure 3. Temperature dependence of the hydrodynamic diameters (D ) of polymerꢀPIR
H
conjugates PC2 - PC4 (solid lines) and their corresponding precursors PP2 – PP4 (dashed lines)
measured by DLS.
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