Application Of Doxorubicin-loaded Poly(Ethylene Glycol)-polyleucine Micelles For The Treatment Of Osteosarcoma

Background:Osteosarcoma (OS) is regarded as the most common primary malignant bonetumor in orthopaedics, mostly locating in the metaphysis of long bones, such as distalfemor, proximal tibia and proximal humerus. Clinically, OS is most prevalent inchildren and young adults, and it results in significant pain, swelling, dysfunction inthe affected limb and relative high mortality. The long-term survival rate of the OSpatients is greatly compromised by the onset of lung metastases.Currently, the standard treatment method of OS associates surgical treatment, andneoadjuvant and adjuvant chemotherapies. However, the current therapeutic strategyis far from perfect because of the shortcomings of poor specificity, drug resistance anddose-dependent toxicity towards normal tissues. Doxorubicin (DOX) plays atremendous role in the multi-agent chemotherapy for OS, but is also recognized as itscardiotoxity and myelosuppression. Various polymeric nanoparticles includingmicelles, vesicles and nanogels are under investigation for the potential usage as drugdelivery system for small molecular weight antitumor drugs. Among these,micellization is considered as an emerging valuable technique to optimize treatmentsowing to its favourable biocompatibility, controlled biodegradation, easy surfacemodification and structure of a hydrophilic shell and a hydrophobic core after theself-assembly of amphiphilic copolymers in aqueous solution. All these advantagesendow the antitumor drugs with the ability to escape from glomerular filtration andcapture of reticuloendothelial system. At the same time, this strategy accomplishes thedemand for enhanced accumulation at the tumor site specifically with thecharacteristics of enhanced permeability and retention effect, and reduced unnecessarysystemic toxicities of conventional antitumor drugs.In this topics, one diblock and two triblock copolymers containing hydrophilic poly(ethylene glycol)(PEG) moieties and hydrophobic polyleucine (PLeu) blockswere synthesized. All the three copolymers manifested the ability of DOXencapsulation after the self-assembly in aqueous solution. We aimed to give acomprehensive illustration for these micelles from various aspects including thepreparations and characterizations of nanoscale particles, DOX encapsulation, in vitroand in vivo antitumor effects, and finally biosecurity.Material and method：(i) The copolymers containing hydrophilic poly(ethylene glycol)(PEG) moietiesand hydrophobic levorotatory or racemic polyleucine (PLeu) blocks were synthesizedby the ring-opening polymerization (ROP) of Leu N-carboxyanhydrides (Leu NCAs)with amino-functionalized PEG (mPEG-NH2or NH2-PEG-NH2) as macroinitiators.The characterizations and biocompatibility of copolymers and micelles wereundertaken by series methods including proton nuclear magnetic resonance spectra,Fourier-transform infrared spectra, gel permeation chromatography, critical micelleconcentrations, transmission electron microscope, dynamic laser scattering andtetrazolium (MTT) assay.(ii) DOX was loaded into micelles through a nanoprecipitation technique. Drugloading content and drug loading efficiency were calculated after the encapsulation ofDOX. The behavior of in vitro DOX release was measured. The intracellular DOXrelease and antitumor effect was detected via confocal laser scanning microscopy(CLSM) and flow cytometry (FCM) detections and MTT assays toward humanosteosarcoma cells, respectively.(iii) We use five-week-old female BALB/c nude mice for xenografted models ofhuman osteosarcoma. Tumor cells were implanted subcutaneously in the armpits, andthe changes in tumor volumes and body weights were real-time documented for theevaluation of chemotherapy efficacy and biosecurity.(iv) Tumors, and major internal organs (i.e., heart, liver, spleen, lung and kidney)were isolated after the whole treatment period of12days. Histopathologicalevaluations were conducted for necrosis, apoptosis of the tumor and systemic toxicity. Results:(i) Diblock copolymer, i.e., mPEG-b-O(D,L-Leu), two triblock copolymers, thatis, P(L-Leu)-b-PEG-b-P(L-Leu) and P(D,L-Leu)-b-PEG-b-P(D,L-Leu) weresynthesized by the ROP of L-Leu NCA, or equivalent L-Leu NCA and D-Leu NCAwith mPEG-NH2or NH2-PEG-NH2as macroinitiator. The chemical structures ofcopolymers were confirmed by the detecting methods mentioned above.(ii) All the three copolymers could self-assemble into micelles in aqueoussolution with the CMCs values at47.8,4.5and3.3mg L-1, and particle diameters of121±4.6,211±5.9and179±7.8nm respectively. The cell vitality at72h was above90%, indicating the ideal biocompatibility. Subsequently, DOX was loaded intomicelles through a nanoprecipitation technique, obtaining the relative high drugloading content at12.0±0.5,18.3±0.4and19.7±0.4wt.%, respectively. All thethree DOX-loaded micelles exhibited controlled in vitro release with different kinetics.Among these, P(D,L-Leu)-b-PEG-b-P(D,L-Leu) micelle showed the greatestadvantage of sustained-release.(iii) Results of CLSM and FCM assays towards two kinds of osteosarcoma cells,i.e., MG63and Saos-2cells, revealed different velocities of endocytosis andintracellular DOX release. The diblock micelle dominated in this section owing to itsstructure of oligomerization. The ability of in vitro antitumor effects of all the threemicelles was superior to those of free DOX.(iv) After being loaded into micelles, the in vivo antitumor effects of DOX weresignificantly enhanced in xenografted models. The loss of body weights due to thesystemic toxicity decreased simultaneously. The histopathological analyses showedincreased necrosis in tumor areas and less signs of injuries to other organs at the sametime.Conclusion：DOX can be loaded into the above three PEG-Pleu micelles, which weresynthesized by ROP and encapsulation through a nanoprecipitation technique withhigh efficiency. The properties including drug loading, in vitro and intracellular payload release, also antitumor effects can be regulated by the factors including thestructures of polypeptides and chirality of involved amino acids. Most importantly, theDOX-loaded micelles showed great advantages for antitumor effects and biosecurity.With convenient preparation, favourable biocompatibility, efficient drug loading anddirectional release, appropriate biodistribution, and excellent antitumor efficacy andsecurity, the fascinating polypeptide drug delivery systems presented great potentialfor clinical chemotherapy of osteosarcoma.