Design of a Novel Drug Delivery Nanosystem that Simultaneously Realizes Real‐Time Tracing and Drug Delivery Across the Blood–Brain Barrier

Acute encephalitis is a brain infection that can harm the nervous system if not recognized and treated promptly. However, the presence of the blood–brain barrier restricts therapeutic agent distribution from the bloodstream to the brain parenchyma, severely restricting effective therapy for this disease. Herein, a novel drug delivery system based on a macrophage (RAW 264.7 cells) artifactual diagnostic and therapeutic nanoparticles (IPD@RAW) drug‐loading approach is presented, which exploits RAW cells' ability to cross the blood–brain barrier and go toward inflammation, and efficiently realizes the targeted enrichment of diagnostic and therapeutic nanoparticles at the site of inflammation in the brain. This nano‐drug‐carrying technology can accurately depict the degree of inflammation in real time for an extended period due to the significant penetration depth and high signal‐to‐noise ratio of near‐infrared (NIR) imaging. Meanwhile, the modified polydopamine can trigger the controlled release of anti‐inflammatory drugs through photothermal conversion under NIR irradiation to reduce the expression of cellular inflammatory factors, such as TNF‐α, IL‐6, and IL‐1β, and alleviate the brain damage due to secretion of this inflammatory factor. As a result, this drug delivery system provides a reliable tool for overcoming the blood–brain barrier to achieve early diagnosis and treatment of acute encephalitis.

DOI: 10.1002/admi.202300738neurological symptoms and complications. [1]Brain inflammation is also an accompanying symptom of certain brain diseases. [2]If acute brain inflammation is not treated promptly, in that case, it may cause neurological symptoms, and cause persistent effects and damage to the nervous system, eventually leading to neurological diseases such as Alzheimer's disease and Parkinson's disease. [3]Therefore, early diagnosis and treatment are necessary for acute brain inflammation to avoid the impact and damage to the nervous system. [4]he blood-brain barrier (BBB) serves as a protective layer between the brain's tissue and the blood arteries of the nervous system.Its purpose is to prevent dangerous chemicals from entering brain tissue and to stabilize the brain's internal environment. [5]The BBB is necessary for healthy brain function but can also prevent medications from reaching the central nervous system. [6]Research has shown that <5% of diagnostic and therapeutic drugs can overcome the BBB, and the slow entry of drugs into the brain leads to inadequate detection accuracy and therapeutic effect. [7]Therefore, it is still difficult to diagnose and treat brain illnesses.A medication delivery system that can cross the blood-brain barrier and target the lesion to treat and monitor the disease process is needed. [8]he development of drug-delivery devices is an important part of acute encephalitis diagnosis and therapy. [9]There are now two types of drug delivery at the site of brain injury: invasive and noninvasive.Direct injection of CSF fluid and transient disruption of the blood-brain barrier, such as with high osmotic pressure solutions or ultrasonography, are examples of invasive procedures. [10]rain-targeted drug design customized to the molecular properties of brain lesions, as well as nanocarrier-mediated drug delivery systems such as nanoparticles, liposomes, dendrites, micelles, and extracellular vesicles, are non-invasive techniques. [11]mmune cells, such as macrophages, have emerged as a unique non-invasive drug carrier for the treatment of brain illnesses, according to current studies. [12]Macrophages have the ability to traverse the blood-brain barrier and have a strong surface recognition function, allowing them to locate and bind to secretions in sites of inflammation, boosting medication Scheme 1.The design and procedure for creating a macrophage-camouflaged diagnosis-therapy-based NIR nano complex, IPD@RAW, and its application in visualizing and treating acute inflammation.1) Polydopamine (PDA)-modified NIR nanoparticles (ICG@NPS) were used for NIR imaging.2) ICG@NPS were loaded with the anti-inflammatory drug dexamethasone (IPD).3) RAW engulfed IPD and created the macrophage-camouflaged NIR drug delivery system, IPD@RAW.4) The drug delivery system was then I.V. injected into infected mice.5) Drug delivery was achieved through immune homing, finally penetrating the blood-brain barrier.6) Dex release and therapy (down-regulating the expression of TNF-, IL-6, and IL-1) were facilitated along with visualization.
accumulation and therapeutic efficacy in these locations. [13]Furthermore, macrophages have a larger drug-carrying capacity, lowering the required drug dose and the likelihood of side effects. [14]actors such as acute brain inflammation visualization and drug monitoring must be taken into consideration when designing a drug delivery system. [15]Computed tomography, [16] single photon emission computed tomography, [17] positron emission tomography, [18] photoacoustic imaging, [19] magnetic resonance imaging, [20] and are being employed to identify acute brain inflammation.Most of these approaches are based on morphological tests and frequently include radiation risks.Among imaging methods, fluorescence imaging is a non-invasive imaging technique that has gradually become one of the reliable methods for diagnosing acute brain inflammation due to its non-radioactive contamination, high sensitivity, simple operation, and ability to provide real-time monitoring of disease progression and drug accumulation information. [21]In recent years, near-infrared fluorescence imaging has been used for deep tissue imaging of brain tissue beneath the skull due to its intense tissue penetration and minimal light damage to biological samples, which provide a realtime and high-precision image of lesion localization guidance for diagnosis and treatment. [22]uring this research, a unique drug delivery system (IPD@RAW) based on macrophages (RAW 264.7 cells, shortly RAW) camouflage was developed, as shown in Scheme 1, allowing for efficient and precise drug delivery and real-time observation of acute inflammation in vivo.In this system, IPD@RAW exhibited exceptional diagnostic and therapeutic capabilities, targeting specific disease sites through the immunological recognition ability of RAW cells.Furthermore, NIR imaging was utilized for precise localization and long-term tracking.Polydopamine (PDA)-modified fluorescence nanoparticles (ICG@NPS) served as NIR light-responsive carriers, loading the anti-inflammatory drug dexamethasone (Dex) for inflammation therapy.IPD@RAW crossed the blood-brain barrier in a mouse model of acute brain inflammation, enriched the lesion site via macrophage chemotaxis, and enabled high signal-to-noise ratio imaging of the brain tissue below the skull.By modifying the radiation intensity of the NIR source, it was possible to extend the residency period of Dex in the brain, significantly enhance its therapeutic effect, and down-regulate the expression of inflammatory factors like TNF-, IL-6, IL-1, and in acute brain inflammation.With this intelligent drug delivery system, our strategy has immense potential for inflammation detection and drug delivery and therapy in brain disease.

Establishment and Characterization of IPD
IPD nanoparticles were created using the procedure described in the experiment portion.ICG@NPS was produced by centrifugal separation after the ICG and NPS were dissolved together in a specific swelling agent for three hours.The PDA may be thought of as a bridge to integrating the Dex and ICG@NPS@PDA by electrostatic contact throughout the experiment process, where PDA would stick to the surface of ICG@NPS via a selfpolymerization with dopamine; Dex would be immobilized onto the surface of ICG@NPS@PDA nanoparticles.SEM was used to examine the morphology of ICG@NPS, ICG@NPS@PDA, and IPD to confirm the manufacturing of NPS.The SEM of ICG@NPS is shown in Figure 1a.As depicted in Figure 1b, ICG@NPS was encased in a single layer of polymer, showing that PDA was present within the polymer shell, proving that ICG@NPS@PDA had been successfully fabricated.In addition, the SEM image (Figure 1c) of IPD illustrated that the adsorbed Dex could not destroy the structure of ICG@NPS@PDA in the acid environment.The dynamic light scattering (DLS) detections of ICG@NPS, ICG@NPS@PDA, and IPD are shown in Figure S1 (Supporting Information), in which PDI were 0.015, 0.041, and 0.043, respectively.The modification of IPD components was determined by UV-vis spectrum, fluorescence spectrum, and zeta potential.The UVvis absorbance peaks were 785 and 242 nm, which belong to ICG and Dex, and there is a strong fluorescence emission at 814 nm (Figure 1d).Zeta potential variation on the ICG@NPS surface also showed that PDA and Dex were successfully adjusted (Figure 1e).The relationship between luminescence intensity and concentration was further determined.It can be seen from Figure 1f that there is an excellent linear relationship and sensitivity between gray values and concentrations of IPD, which laid a foundation for the semi-quantification of cell tracking.

Evaluation of the Photothermal Property of ICG@NPS@PDA
NIR irradiation-induced photothermal conversion of ICG@NPS@PDA played a crucial role in determining the drug's therapeutic effectiveness.Upon exposure to 808 nm NIR light, the photothermal images, as depicted in Figure 2a, illustrated that the temperature of ICG@NPS@PDA increased to 49.4 °C, whereas the temperature of ICG@NPS only reached 27.1 °C after 30 min (Figure 2b).The temperature of ICG@NPS@PDA continued to grow after 15 min of irradiation but reached a stable point, as more extended NIR irradiation could cause thermal damage to living organisms.Thus, the initial 15-min period was used to investigate the photothermal properties of ICG@NPS@PDA and the time required for continuous NIR irradiation to trigger drug release.Figure 2c demonstrates the remarkable photothermal conversion capability of ICG@NPS@PDA, with its temperature change being concentration-dependent.As seen in Figure 2d, the temperature of the ICG@NPS@PDA solution (20 μg mL −1 ) increased as the irradiation power density increased.The dependence of temperature on irradiation power was also observed.Additionally, Figure 2e demonstrates that the temperature profiles after five consecutive heating and cooling periods remained almost identical, indicating that ICG@NPS@PDA had good stability in potential drug delivery applications.
To quantitatively evaluate the photothermal transition capability of ICG@NPS@PDA, a NIR laser (at 1.03 W cm −2 ) was employed to irradiate the ICG@NPS@PDA.The temperature change was recorded 30 min after turning off the laser (Figure S2, Supporting Information).Adding a PDA layer to the surface of ICG@NPS enhanced its photostability and photothermal conversion efficiency, yielding exceptional photothermal performance and stability for prospective applications.These results suggest that ICG@NPS@PDA hybrids are attractive candidates for NIR-stimulated medication release and therapy.

Smart Drug Release
It is critical to assess the drug loading capability and pH/ NIR stimulus-responsive drug release performance of ICG@NPS@PDA hybrid nanoparticles before using them as a drug delivery platform.This study used Dex as a typical The photothermal property of ICG@NPS@PDA.a) Photothermographic images ICG@NPS@PDA and ICG@NPS solutions, under NIR light source with a power density of 1.02 W cm −2 .b) Temperature variations in ANPs and ICG@NPS@PDA in 15 min: quantitative statistics.c) Temperature fluctuations of varied ICG@NPS@PDA concentrations with time gradients.d) Temperature variations of ICG@NPS@PDA at different power densities.e) ICG@NPS@PDA (20 μg mL −1 ) photothermal stability under radiation of 1.03 W cm −2 and natural cooling cycles.f) Dex release ratio of IPD with various NIR irradiation times at different power densities.g) IPD drug release behavior when opening and closing NIR light at different power densities alternately.h) Zeta-potential adjustments of IPD following NIR radiation treatment.
drug to test the drug loading capacity and stimulus-responsive drug release performance of ICG@NPS@PDA.The UV-vis spectra of various dosages of Dex were measured and linearly fitted before establishing the system's drug loading and release behavior under NIR light stimulation (Figures S3 and S4, Supporting Information).By comparing the loading rates of ICG@NPS@PDA (20 μg mL −1 ) to Dex at various pH levels, we discovered that ICG@NPS@PDA showed up a pH dependence on the loading rate and reached a maximum efficiency of 57.10% (11.42 μg mL −1 ) at pH 8.0 (Figure S5, Supporting Information).Besides, as shown in Figure S6 (Supporting Information), the loading rate of ICG@NPS@PDA to Dex was leading at a concentration of 50 μg mL −1 (pH 8.0), and the reaction equilibrium between ICG@NPS@PDA and Dex was achieved at 40 min.The remarkable loading efficiency could be attributed to the electrostatic solid contact between the negatively charged Dex and positively charged PDA.
Dex has good solubility in an acidic environment; thus, IPD nanoparticles were expected to exhibit exceptional pH responsiveness.When Dex-loaded IPD nanoparticles were immersed in PBS (pH 8.0 and 6.0) for 1 h after NIR laser irradiation (1.03 W cm −2 ), the released Dex amounts were 18.32% (2.09 μg mL −1 ) and 41.52% (4.74 μg mL −1 ), as shown in Figure S7 (Supporting Information).In contrast, 35.11% (4.01 μg mL −1 ) of Dex was released in PBS at pH 7.4 after NIR laser irradiation.Dex-loaded ICG@NPS@PDA released more Dex in an acid medium, such as an inflamed or tumor location.As a result of IPD's excellent drug release characteristic in the cell system or biosystem, the NIRresponsive drug release performance of IPD was further examined at pH 7.4.The irradiation time is an essential factor in studying drug release.The drug release rate was 3.87% (0.44 μg mL −1 ) after irradiation for 10 min but reached about 21.82% (2.49 μg mL −1 ) for 100 min when the excitation power density was 0.32 W cm −2 .Another significant aspect influencing the medication release rate was the excitation power density.As the excitation power density rose, the drug release rate significantly increased; for example, when the power was 1.03 W cm −2 , the drug release rate of Dex reached 59.70% (6.82 μg mL −1 ) (Figure 2f).
We estimated the drug release from IPDs under continuously alternating on and off NIR light to explore the response and drug release ability of IPDs to NIR intensity (Figure 2g).The drug release rate of IPD after irradiation for 5 min with 1.03 W cm −2 NIR light was significantly increased and reached 19.4% (2.22 μg mL −1 ), much higher than that of IPD without NIR light irradiation (only 1.2%).After turning off the NIR light source, the drug release rate decreased to about 0.8% (0.09 μg mL −1 ).After the first hour, turning on the NIR light source caused the drug release rate for IPD to quickly climb to 20.4% (2.33 μg mL −1 ), compared to a 1.3% increase in the drug release rate without the NIR light source, indicating reliance on the NIR light source.The drug release rate of IPD increased with increasing power density after 4 h of alternating NIR light sources' activity at various power densities.IPD treated with 1.03 W cm −2 NIR radiation had a more significant drug release rate than the other groups, reaching 47.9% (5.47 μg mL −1 ).The drug release rate of IPD treated with 0.34 W cm −2 NIR radiation reached 16.2% (1.85 μg mL −1 ) in a power density-dependent manner.We discovered that drug release caused changes in IPD surface charge when combined with changes in IPD zeta potential before and after the action of NIR light (Figure 2h).As a result, as an on-demand medication release nanosystem, IPDs could be used to treat inflammation.To acquire the ideal Dex therapeutic concentration, the power density and irradiation time of the NIR source was adjusted, and the Dex release was controlled within a safe concentration range to minimize tissue toxicity.

IPD's Cellular Uptake and Mechanism
To ascertain cellular uptake behavior, ICG, ICG@NPS, ICG@NPS@PDA, and IPD were co-incubated with RAW cells to see how their access to the cells was influenced by cellular uptake.The uptake findings were further disclosed by laser confocal imaging, and fluorescence analysis was carried out using the ICG's near-infrared light signature.As shown in Figure 3a, after 3 h of incubation with ICG, ICG@NPS, ICG@NPS@PDA, and IPD, we detected brilliant fluorescence in the cytoplasm, indicating that both ICG@NPS@PDA and IPD could be taken up by RAW cells.It is also worth noting that ICG@NPS-treated cells had higher fluorescence intensity than ICG@NPS@PDA-treated cells.For assessing the differences in RAW cellular uptake between ICG@NPS@PDA and IPD, fluorescence analysis was used to detect their uptake patterns.The results indicated that the cellular uptake of ICG@NPS@PDA showed a temporal gradient, and the cellular uptake reached the maximum value (48.4%) at 3 h, indicating a time-dependent process of their cellular uptake.The modification of PDA increased the efficiency of ICG@NPS@PDA uptake by cells more than that in the ICG@NPS treatment group (Figure S8, Supporting Information).
Cytotoxicity is a crucial biomarker for assessing the safety of pharmacological nanocarriers.For verifying the biological toxicity of Dex produced by IPD under NIR irradiation to cells, cells were incubated with Dex, IPD, and IPD treated with NIR, then stained with Calcein-AM and PI.The confocal imaging findings in Figure 3b indicated that, compared with all other groups, the IPD therapy group exposed to NIR light showed the most effective performance.Furthermore, the extensively used MTT test was utilized to examine the in vitro cytotoxicity of ICG@NPS@PDA, IPD, and IPD+NIR in RAW cell lines.As shown in Figure 3c, when the concentration reached 160 μg mL −1 , there was almost no cell death after 48 h of incubation with ICG@NPS@PDA and IPD, indicating that ICG@NPS@PDA and IPD had extremely low cytotoxicity and good biocompatibility.In contrast, NIR-treated IPD exhibited some concentration-dependent cytotoxicity.Approximately 27.6% of RAW cells suffered apoptosis following 48 h of exposure to IPD at a dose of 160 μg mL −1 .The findings showed that IPD triggered macrophage apoptotic pathways and induced macrophage apoptosis, in which IPD combined with NIR irradiation dramatically enhanced the effect of IPD on cells.
Through in vitro coincubation, the camouflaged nanocomplex IPD@RAW was produced.The intracellular loading dynamics were tested under different concentrations of IPD over an extended period, as depicted in Figure 3d.Analysis of the loading ratio revealed that IPD was taken up by cells in a time-dependent way, with maximum cell uptake (45.6%) occurring at 2 h when the IPD concentration was 100 μg mL −1 .The uptake rate remained saturated after that.The loading ratio reduced to 19.5% at an IPD concentration of 40 μg mL −1 , showing concentration dependence.Furthermore, as demonstrated in Figure 3e, the retention ratio of IPD in RAW cells remained high (88.6%)after 48 h in DMEM.As a result, IPD@RAW was successfully assembled and had a high potential for long-term monitoring.
Figure 3f illustrates the temperature changes of the IPD@RAW and RAW solution concerning NIR irradiation time.The results showed that the temperature of the IPD@RAW solution was related to the irradiation power density and increased with the enhancement of the power density.For examining the NIR-stimulated drug release efficiency of IPD@RAW further, a laser was turned on, and NIR was irradiated for 15 min at the 2-h point in DMEM.The Dex release curves were recorded and shown in Figure 3g.Dex released increased significantly after the laser was turned on and reached a final value of 33.02%, higher than the value observed without a laser (7.96%).This result demonstrates the remarkable NIR-responsiveness drug release performance of IPD@RAW. Figure 3h shows the Dexreleased curves of IPD@RAW when exposed to varying levels of NIR laser illumination for 20 min.At an intensity of 1.03 W cm −2 for 10 h, almost 36.68% of Dex was released, which was higher than the values recorded at 0.68 W cm −2 (30.01%), 0.42 W cm −2 (22.43%), and 0.34 W cm −2 (11.09%), respectively.The photothermal impact of IPD@RAW, which lessened the interactions between Dex and PDA, is responsible for these NIR laser intensity-dependent drug release characteristics (Figure 3f).

Visualization and Therapy of Acute Brain Inflammation
Acute inflammation can affect other distant organs and signify localized or systemic disease. [23]Consequently, a sensitive and intuitive visualization technique is required.Because of its deep location and cranial occlusion, brain inflammation presents a significant obstacle for optical imaging.Lipopolysaccharide (LPS), a component of the outer membrane of Gram-negative bacteria, [24] was used to create a model of inflammation in a central or peripheral nervous system.IPD@RAW was used to illuminate the inflamed regions of the brain.Based on the fluorescence imaging ability of IPD@RAW, the retention of IPD@RAW in the brains of mice with inflammation models was detected by intravenous injection.As expected, due to the presence of the reticuloendothelial system, IPD@RAW first provided distinct signals in the lungs and liver regions (Figure S9, Supporting Information).After 3 h of medication treatment injection, a significant fluorescence signal was visible in multiple time-lapse photos of the mouse brain, reaching the inflammation tracer at 48 h.At 12 h after injection, the signal-to-noise ratio was up to 105, indicating that RAW can delay the intracerebral lymphatic system's quick drug clearance and extend its duration in the brain (Figure 4aI).The biodistribution of the IPD@RAW preparation was subsequently determined by imaging the ex vivo fluorescence signals of each organ (heart, livers, spleen, lungs, kidneys, and brain).After that, the mice were euthanized 3 h after the injection, and the harvested organs were imaged (Figure S10, Supporting Information).The imaging results showed that IPD@RAW accumulated not only in the livers but also in the brain.Healthy mice in the control group without inflammation induction were also intravenously injected with IPD@RAW, and the brain displayed essentially slight visible fluorescence at all periods (Figure 4aIV), with signals occurring exclusively in the thoracic region (Figure S11, Supporting Information).To test the mechanism by which IPD@RAW lights up the inflamed brain, we also employed PC-3 cell-camouflaging nano complexes (IPD@PC-3) and IPD.The signals in Figure 4a II and III groups only appeared in the thoracic cavity area, and brains were not visible in any group.The average fluorescent signals quantitative analysis of brains and livers are shown in Figure 4b,c.
It was found that the photothermal conversion ability of IPD@RAW in mice under NIR light could impact the release of loaded drugs, which in turn affected the therapeutic outcome of IPD@RAW in a brain inflammation model.For studying the response of IPD@RAW to NIR light and its photothermal conversion capabilities in mice brains, an I.V. injection of IPD@RAW or saline was administered to inflamed mice, and the photothermal effects were analyzed under NIR irradiation.As depicted in Figure 4d,e, there was a significant rise in temperature in the inflamed mice brains injected with IPD@RAW, reaching 42.2 °C after 10 min of NIR radiation.Conversely, the temperature rise in the brains of injection saline was only up to 34.8 °C in the control group.Remarkably, the brain's IPD@RAW could still react to the NIR light by converting its energy into heat, which would then cause the release of the medication.

IPD@RAW's In Vivo Modulation of Inflammatory Factors
To analyze subtle morphological changes in the brain, we analyzed brain tissue sections and changes in the histopathology of brain cells.H&E staining images revealed significant organ lesions in the inflamed mouse brain; however, after treatment with IPD@RAW+NIR, there was a reduction in cell edema.(Figure S12, Supporting Information).
As inflammatory cytokines generated by brain cells play an essential role in developing brain inflammation, brain samples were also exposed to immunohistochemical staining to demonstrate further the efficacy of the RAW cell drug delivery systems.Therefore, five groups' brain samples were collected in order to evaluate the control of IPD@RAW on brain inflammatory factors.Immunohistochemistry was then used to examine the expression of inflammatory components.Following brain inflammation, TNF-, IL-6, and IL-1 excretion rose considerably, further modulating the inflammatory response.IPD@RAW+NIR therapy, on the other hand, dramatically reduced inflammatory cytokine expression (as displayed in Figure 5a).The average fluorescence intensity for TNF-, IL-6, and IL-1 was subjected to semiquantitative analysis, which revealed significant differences between inflammatory mice with and without NIR light irradiation.These results strongly suggest that IPD@RAW treated with NIR radiation has a downregulatory effect on inflammatory factors (Figure S13, Supporting Information).
Additionally, IPD@RAW+NIR effectively inhibited the overexpression of TNF-, IL-6, and IL-1 and induced by inflammation in serum (Figure 5b) and brain tissue homogenate supernatant (Figure 5c), proving that IPD@RAW+NIR was more effective than Dex and IPD@RAW at reducing the secretion level of the targeted inflammatory cytokines.These findings revealed that IPD@RAW could be recruited to cross the BBB and accumulate in the brain inflammatory region due to RAW's homing action and that the released Dex with NIR radiation had an antiinflammatory impact.
The safety of the cell-based drug delivery system in vivo is a crucial factor for its further application.After 7 days of medication, H&E-stained sections of mice's major organs, including the kidney, lung, spleen, heart, and liver, were analyzed to determine the adverse side effects of IPD@RAW on critical organs during in vivo metabolism.Figure 6 shows that the texture of each organ appeared normal, with no obvious pathological signs such as inflammation, edema, or necrosis.As a result, the RAW-based drug delivery system demonstrated good biocompatibility with no toxicity or adverse effects during the body's metabolic process.The NIR-triggered drug release from IPD@RAW loaded with drugs is auspicious in targeting inflamed sites of the brain due to the macrophages' targeting ability.This mechanism keeps the medication concentration within a safe range during metabolism, minimizing hazardous side effects.
Our study shows that using macrophages to phagocytize IPD significantly reduces the risk of particle accumulation and capillary clogging.Additionally, phagocytosis of IPD into macrophages may decrease the nanomaterial's toxicity due to their reduced exposure to blood.In conclusion, combining IPD@RAW and NIR-triggered drug release boasts high biocompatibility and safety.
Figure 6.Toxicity testing in mice.After 7 days of therapy, tissue samples from both the drug-administered group and the control group were stained with H&E using kidney, lung, liver, spleen, and heart tissue sections.NIR radiation power density was 1.03 W cm −2 , and the scale bar indicated 100 μm.

Conclusion
Acute brain inflammation can destroy the nervous system, so it is crucial to establish a targeted diagnosis and treatment method.In this study, we constructed an intelligent drug delivery system IPD@RAW based on RAW cell camouflage strategy, which could cross the blood-brain barrier, target acute inflammation, and realize both treatment and visualization.The NIR nanoparticles used had the characteristics of considerable penetration depth and high signal-to-noise ratio.Through the chemotaxis of RAW cells on inflammation, IPD enriched at the inflammatory site releases Dex through photothermal conversion, thus significantly reducing the secretion levels of inflammatory factors TNF-, IL-1, and IL-6.In addition, in the established model of acute brain inflammation, IPD@RAW showed a solid ability to penetrate the BBB and illuminate the inflammatory area covered by the skull, achieving a 48 h tracer of inflammation and a signal-to-noise ratio of 105 at 12 h of injection, which could be visualized in realtime.Dex triggered by near-infrared could alleviate brain inflammation, and this approach could be employed as a reliable drug delivery tool to identify and treat brain inflammation.The study showed the great potential of macrophages in constructing smart drug delivery systems, providing an effective strategy for monitoring the stage of inflammation deterioration and promoting early diagnosis and treatment of disease in the future.

Experimental Section
Establishment of ICG@NPS: The ICG@NPS were synthesized using the method as follows: 1 mL polystyrene nanoparticles (aqueous solution, 5 mg mL −1 ) and 5 mg of ICG were mixed first, and added 0.5 mL swelling agent acetone for shock reaction 3 h; After that, the fluorescent nanoparticles (ICG@NPS) were then distributed in 5 mL of deionized water and retrieved by gradient centrifugation for usage in further.
ICG@NPS@PDA Preparation: The prepared ICG@NPS solution (1 mL) was mixed with 1 ml Tris-HCl buffer solution (pH 8.5) and treated with ultrasonic for 5 min.Then, 1 ml of dopamine (5 mg, Tris-HCl pH 8.5) was progressively added and carefully mixed for 2 h.Finally, after centrifugation, the fluorescent nanoparticles@polydopamine (ICG@NPS@PDA) were dispersed into deionized water (1 mL) at 4 °C for subsequent usage.
Establishment of IPD: The prepared ICG@NPS@PDA solution (1 mL) was taken ultrasound for 5 min, and 1 mL of Dex (in PBS, the concentration is 1 mg mL −1 ) was added and followed by stirring continuously for 1 h.Then, ICG@NPS@PDA@Dex (IPD) was obtained after centrifugation.Finally, the prepared APD was dispersed in 1 mL PBS and stored at 4 °C for later use.
Photothermal Property Analysis of ANPs@PDA: Under a near-infrared thermographic camera with an 808 nm wavelength, the photothermal imaging of ICG@NPS@PDA and ICG@NPS at various intervals was examined.To investigate the photothermal performance of the compound, sample tubes containing 1 mL of multiple concentrations of ICG@NPS@PDA were vertically irradiated with an NIR light source.The temperature was measured every 30 s, and it rose with longer exposure times.The laser power density was changed to record the temperature of ICG@NPS@PDA at various irradiation times after the NIR camera was used to perform photothermal imaging of the NIR radiation of ICG@NPS@PDA and ICG@NPS at multiple times.The ICG@NPS@PDA solution was exposed to NIR light for 15 min to assess the photothermal stability.The solution was then allowed to cool naturally while the NIR light was turned off, and every 30 s, the temperature was measured.Similarly, the solution underwent five cycles of radiation for 5 min and cooling for 10 min.Every 30 s, the solution's temperature was recorded.ICG@NPS@PDA's photothermal stability was identified.
Drug Loading Ratio of ICG@NPS@PDA: ICG@NPS@PDA dissolved in PBS at various pH levels (6.0, 7.4, and 8.0) were combined with varying concentrations of the Dex solution and swirled for 1 h at room temperature using a magnetic stirrer.Centrifugation was used to separate the unloaded Dex, and PBS was used for several PBS washes.Based on the absorbance of Dex at 242 nm in its UV-vis spectrum throughout a series of reaction times (10, 20, 30, 40, 50, and 60 min), the amount of Dex in the supernatant was calculated.The concentration of Dex was also an important factor for the drug loading ratio, so further the influence of Dex concentrations (25, 50, and 100 μg mL −1 ) on ICG@NPS@PDA drug loading ratio were investigated.The drug-loading ratio was determined using the formula of drug loading ratio (%) = (c 0 V 0 −cV)/c 0 V 0 , where c (mg mL −1 ) and V (mL) represent the concentration and volume of the supernatant of Dex, respectively, and c 0 (mg mL −1 ) and V 0 (mL) stand for the initial concentration and addition volume, respectively, to Dex.
Photothermal-Triggered Drug Release: The IPD solution was vertically illuminated with NIR light at room temperature in PBS with various pH levels and varying power densities.The NIR dependent-Dex release was then assessed by doing the following steps: IPD was exposed to multiple NIR power densities for 10 min.It was then quickly centrifuged at high speed to measure the absorbance through a UV-vis spectrophotometer.The UV-vis absorbance was used to calculate the Dex release ratio.The released Dex was then determined using the abovementioned method after the supernatant was combined with the residue and let to stand for 50 min at room temperature.The process was constantly measured for three hours after the following phase was repeated.Additionally, the Zeta potential of IPD was determined before and after NIR irradiation.
In Vitro Imaging: All luminescence imaging tests were conducted using in vivo imaging equipment.In vitro, imaging was done by putting IPD in a 384-well plate.The fluorescence images were acquired with light source irradiation at 785 nm.Signals were obtained from a fluorescence imaging channel of 815 nm and analyzed with Bruker Molecular Imaging Software and Andor Solis software.
Cell Culture: RAW cells and PC-3 cells were generously donated by The Chinese Academy of Sciences Cell Bank.They were seeded in T25 culture plates and cultured for one day with a DMEM containing 10% (v/v) FBS and 1% antibiotics (100 μg mL −1 streptomycin and 100 U mL −1 penicillin) in a 37 °C CO 2 incubator.
Preparation of Drug Delivery System IPD@RAW: RAW cells were cultured in DMED (without FPS) for 3 h with IPD ((100 μg mL −1 ).After the media was withdrawn, the cells were washed five times using PBS to eliminate any remaining nanoparticles.To create a drug delivery system (IPD@RAW), the cells were trypsin digested and centrifuged.IPD@RAW was then disseminated in PBS solution for subsequent usage.
Cell Activity Assay: The cytotoxicity of IPD, Dex, and IPD with NIR808 was assessed using the MTT assay.In a nutshell, RAW cells were cultured in dishes with 96-well (1 × 10 4 cells per well) and exposed to a range of doses of IPD, Dex, or IPD+NIR for 48 h.The cells were thoroughly rinsed in PBS before incubating at 37 °C for 4 h with 20 μL MTT (in PBS, 0.5 mg mL −1 ).The supernatant was then taken out, and 100 μL of dimethyl sulfoxide was put into each well of the plate.The 96-well plate was gently shaken for 10 min to dissolve the formazan crystal.Then, the absorbance at 490 nm was recorded using a microplate reader.
Calcein-AM / PI staining was used to determine cell survival further.Overnight, RAW cells were planted in a glass bottom dish.ICG@NPS, ICG@NPS@PDA, and IPD were added into the dish and incubated for 48 h at 37 °C.The supernatant was then removed.The dish was then filled with 150 μL of working solution (5 μL Calcein-AM solution (2 mm), 15 μL PI solution (1.5 mm), 5 mL buffer solution) and incubated at 37 °C for 15 min.Finally, the cells were rinsed thoroughly with PBS and used to detect living cells (green fluorescence) and death cells (red fluorescence) via laser confocal microscopy.
Cell Imaging: RAW cells were seeded in a glass bottom dish overnight.ICG@NPS, ICG@NPS@PDA, and IPD (50 μg mL −1 ) were added into the dish and followed with incubation for 4 h at 37 °C.After collecting the supernatant, the cells were carefully washed with PBS.Finally, they were submerged in PBS and imaged using a confocal laser microscope.
Intracellular IPD Loading Dynamics: Overnight seeding of RAW cells in T25 culture plates.The cells were then incubated for 3 h in 5 mL DMEM (without FBS) with 200 μL 50 μg mL −1 IPD, in which the fluorescence spectrum of the cell culture media was monitored every 30 min.The intracellular loading kinetics of IPD were determined by comparing absorbance to a 50 μg mL −1 IPD solution.
Retention Ratio of the APD in RAW Cells: RAW cells were overnight seeded in T25 culture dishes.Following that, the cells were cultured for 3 h with 200 μL 50 μg mL −1 IPD (5 mL DMEM without FBS).The initial value was established as the fluorescence strength of the swallowed IPD.Fresh DMEM without FBS was introduced after the cells had been thoroughly rinsed with PBS.For 48 straight hours, measurements of the fluorescence spectrum of the cell supernatant were taken every 4 h.The fluorescence intensity ratio every four hours to the original value was used to calculate IPD's retention rate in RAW cells.
Study on the Therapeutic Effect of Acute Brain Inflammation Mice: The animal procedures followed the Institutional Animal Care and Use Committee standards in the School of Pharmacy and Pharmaceutical Sciences and Institute of Materia Medica at Shandong First Medical University.Acute brain inflammation was established in the brain inflammation model by intraperitoneal injection of LPS (with a concentration of 10 μg mL −1 ) for 7 days, after which IPD, IPD@RAW (1 × 10 6 cells), and IPD @PC-3 (1 × 10 6 cells) were injected consecutively, and fluorescence images were obtained on the in vivo imaging system.In the control group, the normal mice were I.V. injected with IPD@RAW (1 × 10 6 cells).The accurate localization of the inflammatory site was monitored by fluorescence imaging 3 h later, which was monitored for 48 h and measured every 3 h.Then, the lighted inflammatory area was irradiated with NIR light for 10 min; NIR triggered the release of Dex, which played a therapeutic role at the site of inflammation.Individual tissues were extracted from the euthanized mice on day 10 after varied treatments for further analysis.
Quantifying of Certain Inflammatory Factors by ELISA: After centrifugation, the brain tissue supernatant was collected and homogenized in 1 mL PBS pH 7.4.The quantification of inflammatory factors (TNF-, IL-6, and IL-1) in serum or brain tissues was detected by ELISA kits.The absorbance at 450 nm was measured using a microplate reader and finally got a standard curve.The levels of various inflammatory agents were estimated based on the above-established standard curve.
Semiquantitative Analysis: Semiquantitative analysis of inflammatory factors (such as TNF-, IL-6, and IL-1) from immunohistochemical staining of inflammatory and normal mice, the calculation process was as follows: Immunohistochemical staining luminescence images were recorded on a confocal fluorescence microscope.The target areas were selected on luminescence images, and the gray values were read on Andor Solis software.Then, Origin software and Microsoft Excel were used to draw the histogram and statistical analysis, respectively.
Statistical Analysis: All data were expressed as the standard deviation of the mean of the three independent experiments average value, and the Student's t-test was performed to evaluate whether the differences in group means were statistically significant.When *** p < 0.001, differences across groups were statistically significant.

Figure 1 .
Figure1.Characterization of IPD.FESEM of ICG@NPS a), ICG@NPS@PDA b), and IPD c).d) UV-vis absorbance spectrum (blue) and fluorescence spectrum of IPD (red).e) Zeta potential of ICG@NPS, ICG@NPS@PDA and IPD.f) The average gray values of IPD with different concentrations in each well.

Figure 2 .
Figure2.The photothermal property of ICG@NPS@PDA.a) Photothermographic images ICG@NPS@PDA and ICG@NPS solutions, under NIR light source with a power density of 1.02 W cm −2 .b) Temperature variations in ANPs and ICG@NPS@PDA in 15 min: quantitative statistics.c) Temperature fluctuations of varied ICG@NPS@PDA concentrations with time gradients.d) Temperature variations of ICG@NPS@PDA at different power densities.e) ICG@NPS@PDA (20 μg mL −1 ) photothermal stability under radiation of 1.03 W cm −2 and natural cooling cycles.f) Dex release ratio of IPD with various NIR irradiation times at different power densities.g) IPD drug release behavior when opening and closing NIR light at different power densities alternately.h) Zeta-potential adjustments of IPD following NIR radiation treatment.

Figure 3 .
Figure 3.The behavior of IPD in RAW cells.a) The laser confocal imaging of RAW cells after coincubation with ICG, ICG@NPS, ICG@NPS@PDA, and IPD.b) The imaging of RAW cells after co-incubating Dex, IPD, and NIR-radiated IPD with the Calcein-AM/PI apoptosis kit.The scale bar indicates 50 μm.c) Cytotoxicity assessment of ICG@NPS, IPD, and NIR-radiated IPD.d) and e) The loading rate and retention rate of APD in RAW over time.f) Temperature changes of APD@RAW and RAW solutions under the NIR light irradiation (the power density was 1.03 W cm −2 ) g) Photo-triggered Dex release from APD@RAW with and without NIR laser irradiation.h) Dex release curves of IPD@RAW with NIR irradiation at different power densities.Error bars represent the standard deviation of the mean, while all data represent the average of n determinations (n = 3).

Figure 4 .
Figure 4. Visualization and photothermal effect study of acute brain inflammation.a) Fluorescence imaging at various time periods following I.V. injections of IPD@RAW, IPD, IPD@PC-3, and IPD@RAW into inflamed mice and IPD@RAW into normal mice.The scale bar indicated 1 cm.Average liver and brain fluorescence intensities b,c, respectively) as a function of post-injection time in (a).d) Following intravenous injections of IPD@RAW or saline, temperature fluctuations and thermal imaging of the brain were observed under NIR808 light with a NIR laser power density of 1.03 W cm −2 .The scale bar indicated 1 cm.e) Statistical analysis of brain photothermal images.

Figure 5 .
Figure 5.The effect of IPD@RAW+NIR on inflammatory factors in mouse brain tissue.a) TNF-, IL-6, and IL-1 immunohistochemical staining in brain extracts from inflammatory mice with and without NIR irradiation, as well as normal mice.The scale bar indicates 50 μm.Investigation of the effects of IPD@RAW+NIR on inflammatory factors.Quantitative analysis of certain inflammatory factors (TNF-, IL-6, and IL-1) in serum b) and brain tissue homogenate supernatant c) after Dex, IPD@RAW, and IPD@RAW+NIR treatment.Where *** p < 0.001.