Construction Of Cartilage Degeneration-Targeted Probe And Its Application In Photoacoustic Imaging Of Early Osteoarthritis

Part I The construction,characterization and identification of melaninnanoparticles photoacoustic molecular probe.Objective In vivo photoacoustic imaging(PAI)is a non-invasive and non ionizing imaging modality developed in recent years.It possesses high resolution and contrast,and has been widely used in the diagnosis of small animal disease model.However,in the pathophysiology of most diseases,there is a lack of endogenous PA agent that can reflect pathological changes.Here,we designed a PA molecular probe for natural melanin nanoparticles(MNPs)modified by poly-L-Lysine(PLL),which is rich in positive charge can specifically bind to glycosaminoglycans(GAGs)in articular cartilage,and to quantify the changes of GAG content during cartilage degeneration.The first part of the experiment is to prepare nano PA probes PLL-MNPs,and evaluate the characterization and identification of the PA probes.Methods Preparation of water soluble melanin nanoparticles:The melanin particles were dissolved in NaOH aqueous solution of 0.1N,and the bright aqueous solution of melanin was obtained under ultrasonic(output power =10W)oscillation for 1 minutes.The solution was modulated with its pH value to neutral(PH=7)and filtered.Then removed the sodium with deionized water,and the solution was solvent by a freeze dryer to obtain ultrafine MNPs.Enveloped with PLL:PLL was added into the prepared MNP aqueous solution,stirred and mixed.Through a series of chemical reaction,MNP was enveloped by PLL.Characterization and identification of PLL-MNPs nanoparticles:Testing water solubility and stability in PBS;UV absorption spectra were measured in tetrahydrofuran;measurement of blood stability in calf serum;potential analyzer for measuring the Zeta potential;light stability of laser irradiated probe;PA characteristics of probe measured by PAI system;and evaluated the cytotoxicity of PLL-MNPs by MTT.Results We successfully synthesized the nano photoacoustic probe PLL-MNPs.The nanoparticles are brownish black and have good water solubility.The solubility can reach 5mg/mL,and the hydrated particle size is stable 42.5 + 1.6 nm.The can be stored in aqueous solution for a long time without aggregation and precipitation.The near-infrared absorption of PLL-MNPs photoacoustic probe is wider in aqueous solution.In this experiment,we take 680 nm as the wavelength of photoacoustic excitation.The Zeta potential of the probe was-22.2 +12.1 MV.After 680 nm wavelength laser irradiation(8mJ Ccm-2),the absorption spectrum of PLL-MNPs solution in UV Vis near infrared spectroscopy showed that the absorption was almost not weakened,indicating that the PLL-MNPs had good photostability.Cytotoxicity test showed that the viability was above 90%after incubation for 24 and 48 hours with different concentrations of PLL-MNPs.Conclusions We have successfully prepared photoacoustic molecular probe PLL-MNPs.It was easily fabricated,biocompatible,and had good PA intensity.These advantages make it possible for further application in vivo.Part Ⅱ Evaluation of targeting ability,sensitivity and specificity of PLL-MNPs in cartilage in vitroObjective To investigate the sensitivity and specificity of PLL-MNPs in targeting GAGs binding ability of cartilage in vitro,and the ability of monitoring the changes of GAGs content in cartilage degeneration.Methods The targeting specificity of PLL-MNPs to GAGs in cartilage was evaluated in vitro by uptake,binding affinity and retention ability experiments.For uptake,all normal cartilage samples(5mg wet weight from 10-week-old SD rats)were incubated with PLL-MNPs at a concentration of 0.5 mg/ml.Samples were removed at different time points(0-72h)after immersion,washed with cold PBS,and imaged using a PAI system by pulsed laser excitation at 680nmin an EP tube.For comparison,MNPs and PBS were subjected to the same procedure.To further confirm that PLL-MNPs efficiently bind to GAGs by electrostatic attraction in articular cartilage,we performed a competitive experiment using PLL as a cationic agent to compete with PLL-MNPs to bind to articular cartilage.To investigate the retention ability of the cationic PLL-MNPs in articular cartilage,cartilage samples were incubated with PLL-MNPs and MNPs for 24 h and immersed in PBS reservoirs at room temperature.At different immersion time points(0-72 h),cartilage samples were removed and imaged by PAI.To investigate the feasibility of PLL-MNPs for PAI of cartilage degradation,a correlation analysis between GAG content and PA agent uptake was conducted in the articular cartilage.Cartilage samples were treated with Chondroitinase ABC at different concentrations to obtain a range of degraded cartilage GAG contents.Following degradation,samples were incubated with PLL-MNPs and MNPs respectively for 24 h.PAI was conducted according to the above in vitro protocol.Results Uptake experiments showed that PLL-MNPs with positive charge had stronger ability of cartilage uptake.The PA signal of the original cartilage was 200.After 24 hours of incubation with the probes,PLL-MNPs showed a stronger PA signal intensity(2580 + 83)than that of the MNPs group(1425 + 46).The ability of PLL-MNPs uptake in cartilage decreased significantly after pretreatment with blocking agent,demonstrating the binding ability of cationic PA probe to negatively charged GAG in cartilage.In the retention experiment,cationic PLL-MNPs exhibited significantly higher PA intensity over 48 hours after immersion,and over 50%of the PA signal remained detectable at 24 h.The PA signal ratio of the PLL-MNPs to MNPs was time-dependent,reaching the highest value of 1.86 at 24 h after immersion(p<0.01,Figure 4e),indicating that optimizing detection time is important for the best PAI effect.In the degradation analysis,cationic PLL-MNPs aggregated in the cartilage extracellular matrix and presented a positive linear correlation of PA intensity with GAG content(R2 = 0.83,p<0.001).In the MNPs group,little correlation between PA intensity and GAG content was presented(R2 = 0.38,p>0.01).Conclusions Based on the above studies,we determined that positively charged PLL-MNPs possess stronger affinity to GAGs and slower diffusion rates from the articular cartilage,which is good for studying PAI on cartilage degradation.The significant difference in PAI among the different GAG contents illustrates the potential use of PLL-MNPs to investigate cartilage degeneration in vivo.PartⅢ PLL-MNPs as efficient photoacoustic agents for early diagnosis of articular cartilage degeneration of osteoarthritis in living miceObjective To verify the effectiveness of PLL-MNPs in the early diagnosis of cartilage degeneration of OA in vivo.Methods Surgical destabilization of the medial meniscus(DMM)was applied to model OA in the right knee joint.Mice(n = 5)were injected intra-articularly(IA)with 10 μg of PLL-MNPs(0.5 mg/ml)at 4 weeks post-surgery,and the other two groups were injected with MNPs and PBS as controls.PAIs were acquired at different injection time points by pulsed laser excitation at 680 nm.To further verify the advantages of PAI using PLL-MNPs for cartilage degeneration,X-rays and MRI(T1 and T2-weighted)were performed on the same mice(4 weeks post-surgery).To assess the feasibility of PAI using PLL-MNPs to differentiate early OA from late OA in vivo,mice were divided into three groups:the normal group,early OA group(4 weeks post-surgery)and late OA group(8 weeks post-surgery).Pathological examination of model specimens was performed at the end of each phase.Results For the first time,the photoacoustic imaging system has been used to achieve clear anatomical imaging of the knee joint.The early osteoarthritis model was successfully established by DMM.Quantitative analysis shows that only a weak increase in PA intensity was observed in OA joints at 3 h post injection,which is significantly lower than in the normal knee joint(1441 ± 109 vs.2113 ± 103,p<0.01).Additionally,the PLL-MNPs retention half-life in OA joints was 22 h,which was significantly shorter than in the normal joints at 48 hours(p<0.01).In comparison,the MNPs and PBS groups did not significantly differ in PA intensity between the normal and OA joints at any time point post-injection(p>0.05).Compared with X-rays and MRI,PAI using PLL-MNPs detects biochemical changes indicative of cartilage degeneration,allowing for a true early diagnosis of cartilage degeneration.The PA signal intensity was significantly higher in early OA(1454±127)than in late OA(1036 ± 55)3 h after PLL-MNPs injection(p<0.01,n = 3),indicating that early-stage GAG content is higher than in late-stage OA.Further detection of the toxicity of the probe showed that the probe had no obvious toxicity to the local joints and the main organs.Conclusions In vivo results revealed that the PLL-MNPs enhanced PAI and provided accurate information on cartilage degeneration as well as effectively distinguished early-stage from late-stage cartilage degeneration.Given these merits,the high PLL-MNPs performance has great potential for early diagnosis of cartilage degeneration,suggesting its future use in monitoring cartilage therapy and in PAI-guided cartilage-repairing arthroscopic surgery.