Study On Novel Targeted Drug Delivery Systems Of Homostructured Peptides-based Nanoassemblies And PH-responsive Nanoparticles

In the past decades, studies clearly show that targeted drug delivery systems based onnanoparticles (NPs) have a distinct advantage in drug therapy. Although the existing studieshave made some achievements on overcoming some physiological barriers and realizingtargeted delivery of different drugs, many challenges still exist. On the one hand, currentlyonly a few polymer nanoparticles based drug release systems have been approved forclinical applications. The complexity and uncontrollability of preparation of polymernanomedicines are the main factors that limit the translation of these laboratory findings tothe clinic practice. On the other hand, because pinpointing the location by stimulating theresponsive nanoparticulate delivery systems targeted at the lesion microenvironment is notprecise, the construction of nanosystems with good biocompatibility remains a problem.Our project is going to solve the above mentioned questions through two parts of studies. Inthe first part of study, a simple method was employed to synthesize homostructuredpolypeptides. These polypeptides can self-assemble to form nanoparticles whose structure,particle size, and size distribution are controllable. This type of nanoparticles can serve asthe drug carrier, and can be used to treat arthritis by oral administration of ananti-inflammatory drug indomethacin (IND). In the second study, we prepared a series ofpH-responsive α-cyclodextrin (α-CD) materials by kinetically controlled acetalation toconstruct pH-responsive nanoparticles. These nanoparticles are able to target the acidicmicroenvironment of tumor tissues. Through a series of experiments in vitro and in vivo,the application of these nanocarriers for the delivery of paclitaxel (PTX) was fullyinvestigated for targeted tumor therapy.Methods1. Synthesis of carrier materialsPolymerization of N-carboxy anhydrides of β-benzyl L-aspartate, γ-benzoy glutamicacid, and ε-carbobenzoxy-L-lysine, initiated by1,1,1,3,3,3hexamethyldisilazane, polypeptides1,8, and9could be synthesized. In the existence of n-butylamine,n-hexylamine, and n-octylamine, polypeptides2,3, and4would be synthesized by theaminolysis of peptide1. For the preparation of polypeptides5,6, and7,poly(D,L-succinimide) was synthesized by polymerization of D,L-aspartate in85%H3PO4.Then, n-butylamine, n-hexylamine, and n-octylamine was added to produce peptides5,6,and7, respectively.Acetalated α-cyclodextrin was synthesized by acetalation of α-cCD in the presence ofpyridinium p-toluenesulfonate and2-methoxypropene. Different materials were obtained bycollecting and precipitating reaction mixture at varied time points. The acetalation ofdextran in the presence of pyridinium p-toluenesulfonate and2-methoxypropene gave riseto acetalated dextran (Ac-Dex). Poly(cyclohexane-1,4-diyl acetone dimethylene ketal)(PCADK) was synthesized by condensation reaction of1,4-cyclohexanedimethanol, in thepresence of2.2-dimethoxypropane and p-toluenesulfonic acid.2. Materials characterizationThe material structure was determined by FT-IR and NMR. The molecular structureand molecular weight of various peptides were characterized by MALDI-ToF massspectroscopy. The molecular weight of Ac-Dex and PCADK was determined by gelpermeation chromatography (GPC).3. Preparation of nanoparticlesThe nanoassemblies of various peptides and drug loaded nanoparticles based onpeptide1were prepared by dialysis method. To this end, peptide or peptide/drug wasdissolved in organic solvent which was then removed by dialysis. Ac-aCD nanoparticleswere produced by emulsion method. First, Ac-aCD or Ac-aCD/PTX was dissolved indichloromethane and the solution was emulsified into1%polyvinyl alcohol (PVA). Theobtained oil-in-water (o/w) emulsion was added into0.3wt.%PVA aqueous solution. Thisemulsion was stirred magnetically at room temperature for3h. Nanoparticles werecollected by centrifugation and washed with deionized water. Similar method was utilizedto fabricate PTX nanomedicines based on PLGA, Ac-Dex, or PCADK.4. Drug loading and loading efficiencyIND/peptide-1nanoassemblies were weighed precisely, then the drug was extractedthree times by using3ml ethanol. The extracts were combined and the volume was set to 10ml. Then UV absorbance at310nm was determined by a ultraviolet spectrophotometer.To measure the PTX content, PTX/Ac-aCD nanoparticles were weighed precisely anddissolved in DMSO, PTX concentration was determined by high performance liquidchromatography.5. Characterization of various nanoparticlesMorphology of nanoparticles was characterized by scanning electron microscopy(SEM) and transmission electron microscopy (TEM). Particle size and size distribution wasmeasured by dynamic light scattering (DLS).6. Stability of polypeptide nanoparticlesBlank polypeptide nanoparticles were put into PBS (pH7.4) with trypsin orchymotrypsin. Nanoparticles hydrolysis was observed at different time points, and theirmorphology was characterized by SEM and TEM.7. Hydrolysis of Ac-aCD nanoparticlesAc-aCD nanoparticles were put into PBS with pH7.4or pH5.0. The hydrolysis ofnanoparticles was observed at various time points and digital photos were taken. Changesin the light scattering intensity of nanoparticle colloid solution were determined by DLS.8. In vitro releaseFor in vitro release test, IND/peptide-1nanoassemblies was placed into dialysis tubing,which was immerged into30ml of PBS, IND was set as control. In vitro releaseexperiments were also performed to simulate gastrointestinal (GI) conditions. For thispurpose,0.1M HCl (pH1.2) was employed as the release medium within the first2h,which was then switched into0.01M PBS (pH7.4). The drug release of PTX/Ac-aCD wascarried out under mildly acidic (pH5) or physiological conditions (pH7.4).9. Cytotoxicity evaluationTumor cells and normal cells were cultured with medium containing blank NPs. Thecell proliferation was determined by MTT method. The influence of Ac-aCD NPs on cellapoptosis was evaluated by flow cytometry.10. In vitro antitumor activity of PTX nanoparticlesVarious tumor cells were cultured with medium containing PTX/Ac-aCD NPs,PTX/PLGA NPs, PTX/Ac-Dex NPs, PTX/PCADK NPs, and PTX. The cell viability wasmeasured by MTT method. The influence of various nanomedicines on cell apoptosis was determined by flow cytometry. Cells were cultured with medium containing PTX/Ac-aCDNPs or PTX, then the influence of various treatments on microtubules of various cells wasobserved at various time points.11. Pharmacokinetic study of IND/peptide-1nanoassembliesIND/peptide-1and IND were orally administered via gastric gavage at4mg/kg formale SD rats. Blood samples were collected at specific time points post-dose.12. Pharmacodynamic evaluation on IND/peptide-1nanoparticlesIND/peptide-1, raw IND, blank peptide-1NPs and saline were administered via gastricgavage for male SD rats, After30min of administration, the animals received anintradermal injection of carrageenan solution in the right hindpaw. The right-back pawvolume was measured by a transducer-linked plethysmometer at predetermined timeintervals after carrageenan injection.13. Acute toxicity evaluation of Ac-aCD nanoparticlesMale kunming mice were administered via i.v. injection of blank Ac-aCD180NPssolution at different doses. Mice were weighed and their behaviors were observed for anysigns of illness each day. After15days, animals were sacrificed by cervical dislocationafter anaesthesia. Blood samples were collected for hematological analysis. Organsincluding heart, liver, kidney, lung, and spleen were harvested and weighed.14. In vivo antitumor study on PTX/Ac-aCD nanoparticlesThe B16F10melanoma xenografts were established by subcutaneous inoculation onthe back with a fragment of B16F10tumor for nude mice. After7days, a singleadministration of various formulations was performed by injection via tail vein, while lower(1.1mg/kg), medium (3.3mg/kg), and higher (10mg/kg) doses were administered in thecase of PTX/Ac-aCD NPs. The tumor size and body weight were measured at various timepoints. Saline and PTX were set as control and the dosage of PTX was10mg/kg. In anotherexperiment, in vivo antitumor activity of PTX nanomedicines based on various materialssuch as Ac-Dex, PCADK, PLGA, and Ac-aCD were compared. Administration of variousformulations was performed by injection via tail vein at the dosage of10mg/kg.15. Targeting studyThe paw edema of male SD rats were induced by intradermal injection of carrageenansolution. When obvious swelling occurred on the toes, peptide-1nanoparticles with (CdSe)ZnS quantum dot and saline were administered via gastric gavage for male SD rats.The organs and paws were resected12h after administration of fluorescent nanoparticles.Fresh medium containing Cy5-labeled Ac-aCD NPs incubated cells for different times.Confocal laser scanning microscopy observation was performed by a fluorescencemicroscope for harvested cells.(CdSe)ZnS quantum dots was loaded into Ac-aCDnanoparticles which were injected into tumor-bearing mice via the tail vein injection. Thefluorescence distribution was observed12h after administration in the body of the mice.Results1. Results of project11) Characterization by FT-IR, NMR, and MALDI-ToF mass spectrometry indicates thesuccessful synthesis of polypeptides with different structures and molecular weights.2) Peptide assemblies exhibited well-defined structures with size between60-200nm.When DMSO was used as the solvent, the size distribution of nanoparticle is more uniform.3) The test of cell proliferation shows that peptide-1nanoparticles have no significantcytotoxicity. After incubation in pH7.4PBS and PBS with trypsin or chymotrypsin, noobvious hydrolysis occurred for peptide-1nanoparticles.4) The drug loading content and entrapment efficiency of IND-loaded peptide-1nanospheres is19.8wt.%and98.8%, respectively. Characterization by DSC indicatesmolecular dispersion of drug molecules, since no endothermal peaks could be detected.5) In vitro release suggests that IND/peptide-1nanospheres and IND have no release atpH1.2, while fast release occurred at pH7.4. The release rate of the former is higher thanthat of the latter.6) Pharmacokinetic study shows that IND/peptide-1nanospheres release is faster thanraw IND in SD rats, reaching the peak faster and sustaining high blood drug concentrationfor a longer time. The AUC of nanoparticles was about1.7times of that of raw IND.7) Treatment with IND/peptide-1nanospheres shows strikingly strongeranti-inflammatory efficacy, when compared with raw IND intervention.8) Targeting study reveals that peptide-1nanospheres could accumulate at theinfection site.2. Results of project21) The analysis of FT-IR and NMR shows that Ac-aCD is obtained after reaction. The formation of acetal groups occurred in a few minutes.2) Ac-aCD nanospheres show the diameter of about250nm, which were prepared byemulsion technique. The observation by SEM and TEM shows a well-performed sphericalshape.3) The drug loading content of PTX/Ac-aCD NPs is about10%, which is higher thanthat of nanoparticles based on PLGA, Ac-Dex, or PCADK.4) The hydrolysis rate of Ac-aCD nanospheres in pH5.0PBS is higher than that in pH7.4.5) In vitro release results suggested that the release rate of PTX in pH5.0PBS ishigher than that in pH7.4.6) Cytotoxicity evaluation suggests blank Ac-aCD nanoparticles displayed low toxicity.Compared with the raw PTX, nanoformulations of PTX/PLGA, PTX/Ac-Dex,PTX/PCADK, and PTX/Ac-aCD may increase the activity of PTX. PTX/Ac-aCD NPs showthe strongest activity, which is more obvious to multidrug resistance cells. PTX/Ac-aCDnanoparticles can also facilitate the apopotic process.7) Ac-aCD nanoparticles have no obvious acute toxicity to mice when the dose islower than250mg/kg. The LD50was higher than500mg/kg.8) Ac-aCD nanoparticles can be endocytosed by various tumor cells, and delivered byendosome and lysosome. In addition, nanoparticles can be accumulated in the tumor tissue.9) PTX/Ac-aCD nanoparticles have better in vivo antitumor efficacy and lower sideeffects as compared to PTX, PTX/PLGA nanoparticles, PTX/Ac-Dex nanoparticles, andPTX/PCADK nanoparticles.Conclusion1. Conclusion of project11) Different polypeptides with simple structure were synthetized through a simplemethod, by using β-benzyl L-aspartic acid, glutamic acid, γ-benzyl lutamate,ε-(benzyloxycarbonyl)-L-lysine, and D,L-aspartic acid.2) The synthesized peptides may assemble to form nanoparticles with uniformdistribution and high stability. Peptide-1nanospheres show good biocompatibility.3) Self-assembled IND/peptides-1nanoparticles perform well in drug loading and areable to facilitate drug release, leading to higher dissolution rate and better bioavailability. Self-assembled nanoparticles containing IND obviously reduce the stimulation ofgastrointestinal tracts and may target disease sites. Therefore, they may serve as novelnanocarrier for targeted delivery of various drugs.2．Conclusion of project21) Ac-aCD materials with pH sensitivity were synthetized through simple acetalation.Besides, size-controllable nanoparticles with uniform size distribution are successfullymade by emulsion technique which are highly biocompatible. All NPs based on variousAc-aCDs show pH sensitivity. The hydrolysis rate in weak acid environment is higher thanthat in a neutral environment. The hydrolysis rate of Ac-aCD nanospheres in pH5.0PBS ishigher than that in pH7.4.3) As the cellular endosome and lysosome as well as tumor tissues have acomparatively low pH value, Ac-aCD nanoparticles with pH sensitivity can be delivered tothe endosome/lysosome and tumor tissues.4) While loaded with PTX, Ac-aCD nanoparticles not only show a better performancein drug loading, but also a great advantage in inhibiting tumor growth and accelerating itsapoptosis, as compared to that of PLGA NPs. Ac-aCD nanoparticles may reverse themultidrug resistance. Compared with PTX, PTX/PLGA NPs, PTX/Ac-Dex NPs, andPTX/PCADK NPs, PTX/Ac-aCD nanoparticles showed a much better performance in termsof in vivo antitumor efficacy. Therefore, they may serve as nanocarrier for targeting tumormicroenvironment.