Open AccessCCS ChemistryRESEARCH ARTICLE1 Jan 2022An Exceptional Broad-Spectrum Nanobiocide for Multimodal and Synergistic Inactivation of Drug-Resistant Bacteria Ke Xue†, Cuihong Yang†, Chun Wang, Yang Liu, Jianfeng Liu, Linqi Shi and Chunlei Zhu Ke Xue† Key Laboratory of Functional Polymer Materials of Ministry of Education, State Key Laboratory of Medicinal Chemical Biology, Institute of Polymer Chemistry, College of Chemistry, Nankai University, Tianjin 300071 †K. Xue and C. Yang contributed equally to this work.Google Scholar More articles by this author , Cuihong Yang† Tianjin Key Laboratory of Radiation Medicine and Molecular Nuclear Medicine, Institute of Radiation Medicine, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin 300192 †K. Xue and C. Yang contributed equally to this work.Google Scholar More articles by this author , Chun Wang Key Laboratory of Functional Polymer Materials of Ministry of Education, State Key Laboratory of Medicinal Chemical Biology, Institute of Polymer Chemistry, College of Chemistry, Nankai University, Tianjin 300071 Google Scholar More articles by this author , Yang Liu Key Laboratory of Functional Polymer Materials of Ministry of Education, State Key Laboratory of Medicinal Chemical Biology, Institute of Polymer Chemistry, College of Chemistry, Nankai University, Tianjin 300071 Google Scholar More articles by this author , Jianfeng Liu *Corresponding authors: E-mail Address: [email protected] E-mail Address: [email protected] E-mail Address: [email protected] Tianjin Key Laboratory of Radiation Medicine and Molecular Nuclear Medicine, Institute of Radiation Medicine, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin 300192 Google Scholar More articles by this author , Linqi Shi *Corresponding authors: E-mail Address: [email protected] E-mail Address: [email protected] E-mail Address: [email protected] Key Laboratory of Functional Polymer Materials of Ministry of Education, State Key Laboratory of Medicinal Chemical Biology, Institute of Polymer Chemistry, College of Chemistry, Nankai University, Tianjin 300071 Google Scholar More articles by this author and Chunlei Zhu *Corresponding authors: E-mail Address: [email protected] E-mail Address: [email protected] E-mail Address: [email protected] Key Laboratory of Functional Polymer Materials of Ministry of Education, State Key Laboratory of Medicinal Chemical Biology, Institute of Polymer Chemistry, College of Chemistry, Nankai University, Tianjin 300071 Google Scholar More articles by this author https://doi.org/10.31635/ccschem.021.202000714 SectionsSupplemental MaterialAboutAbstractPDF ToolsAdd to favoritesDownload CitationsTrack Citations ShareFacebookTwitterLinked InEmail This study reports the fabrication of a novel photothermal material formed via the physical blending of excess lauric acid (LA) and cupric acetate, followed by efficient ligand exchange. Surprisingly, the copper–LA complex exhibited a 12-fold enhancement of the molar extinction coefficient in the near-infrared (NIR) region relative to aqueous cupric acetate. Inspired by this interesting finding, we formulated these photothermal materials into colloidally dispersed nanoparticles via a technique that combined nanoprecipitation and in situ surface polymerization for antibacterial studies. The resultant nanoparticles exhibited rapid and stable photothermal responses to NIR irradiation, with a 4-fold enhanced photothermal conversion efficiency relative to aqueous cupric acetate. Since a positively charged monomer was incorporated during in situ surface polymerization, these positively charged nanoparticles were ingested efficiently and subsequently digested by drug-resistant bacteria. By combining the LA-mediated membrane-damaging effect, copper-mediated Fenton-like reaction, as well as the photothermal effect of the copper–LA complex, a broad-spectrum, multimodal, and synergistic antibacterial effect was achieved both in vitro and in vivo, with the killing efficiency up to 99.99% for ampicillin-resistant Escherichia coli (AmprE. coli) and 99.9999% for methicillin-resistant Staphylococcus aureus (MRSA). Our newly developed nanobiocide represents a class of exceptional broad-spectrum antibacterial materials, holding great potential for treating drug-resistant infections in clinical settings. Download figure Download PowerPoint Introduction The overuse and misuse of antibiotics accelerate the emergence of antibiotic resistance, which poses a serious threat to global health.1,2 It is estimated that 700,000 deaths per year are ascribed to drug-resistant bacterial infections worldwide.3,4 Despite a multitude of antibiotic candidates tested, only a few of them have been brought to the market in the last decades. Consequently, it is urgent to develop alternative antibacterial approaches for the treatment of drug-resistant bacterial infections.5–12 Saturated fatty acids are a class of promising biomaterials due to their low-cost, biocompatibility, and biodegradability.13,14 Among them, lauric acid (LA), a medium-chain fatty acid commonly found in coconut palm and milk, is reported to have selective bactericidal activity against Gram-positive bacteria by exerting a membrane-damaging effect.15–17 However, saturated fatty acids are typically difficult to be formulated into colloidally dispersed nanoparticles due to their high crystallinity and poor dispersity.13,18 Although it has been confirmed that the stabilization of fatty-acid nanoparticles with biocompatible surfactants is an effective strategy,19,20 the long alkyl chains in these surfactants are presumed to interact with the interior fatty acids and the residing components. Such interaction would partially impair their physicochemical properties, resulting in diminished material performance. As such, it is imperative to develop a straightforward and feasible method to fabricate colloidally dispersed fatty-acid nanoparticles without sacrificing their physicochemical properties. Despite the strong antimicrobial activity of LA against a variety of Gram-positive bacteria, it is relatively impotent in fighting against Gram-negative bacteria.16,17 Moreover, its bactericidal activity still needs to be improved by integration with other advanced strategies less likely to induce drug resistance. Owing to the deep tissue penetration, as well as the minimal invasiveness, near-infrared (NIR) light-enabled photothermal therapy (PTT) has been extensively explored in the field of antibacterial therapy.6,21–23 In a typical process, the photothermal agents absorb the NIR light and convert it into thermal energy via nonradiative decay pathways, causing local hyperthermia at the target region. Local high temperature leads to irreversible damage by loosening cell membranes and denaturing bacterial proteins, ultimately resulting in pathogenic bacterial inactivation.6,21,24–26 PTT offers a broad-spectrum antibacterial activity against both Gram-positive and Gram-negative bacteria without inducing marked drug resistance.27 Considering all these benefits, various photothermal antibacterial agents have been developed to boost PTT, such as gold-based nanomaterials,28,29 copper-based chalcogenide nanomaterials,30 carbon-based nanomaterials,31 polymer nanoparticles,32–34 and black phosphorus.35 Among them, copper-based materials have attracted much attention due to their low-cost and stable photothermal conversion performance.30,36 It is widely reported that copper salts possess intrinsic antibacterial and antiviral capabilities, presumably due to the generation of reactive oxygen species (ROS) via Fenton-like reactions.37,38 However, the direct use of water-soluble copper salts is infeasible due to their poor photothermal effect and high toxicity to healthy tissues.39 As such, it is highly desired to reduce the adverse impacts of copper ions by forming a water-insoluble copper complex and concurrently endow the resultant complex with a strong photothermal effect to achieve multimodal killing of drug-resistant bacteria. In an experiment, we found that when LA was dissolved in toluene, it was able to efficiently extract copper ions from aqueous cupric acetate by forming a copper–LA complex with a greenish-blue color. More importantly, the molar extinction coefficient (ɛ) of the resultant complex exhibited a 12-fold enhancement relative to that of aqueous cupric acetate (18 M−1 cm−1). Based on such interesting findings, we characterized the photothermal properties of a series of cupric acetate-doped LA solids fabricated by physical blending of excess LA and cupric acetate, followed by efficient ligand exchange. To formulate these photothermal materials into colloidally dispersed nanoparticles for antibacterial studies, the nanoprecipitation method was exquisitely combined with in situ surface polymerization (Scheme 1a).40–44 In situ surface polymerization generated a layer of hydrophilic polymer network on the surface of the nanoparticles obtained from nanoprecipitation, which acted as a protective sheath to prevent the resultant nanoparticles from fusing into large aggregates. As anticipated, the resulting nanoparticles (denoted as Poly-Cu nanoparticles) exhibited a rapid and stable photothermal response to 808-nm NIR irradiation, with a photothermal conversion efficiency (η) of 35.4% (4-fold relative to aqueous cupric acetate). Interestingly, both Gram-negative and Gram-positive drug-resistant bacteria were able to actively ingest the resultant nanoparticles due to the net-positive charge on the surface of Poly-Cu nanoparticles. By combining the strong photothermal effect of the copper–LA complex as well as the membrane-damaging effect and Fenton-like reactions mediated by the released LA and copper–LA complex, respectively (Scheme 1b), Poly-Cu nanoparticles imposed a broad-spectrum, multimodal, and synergistic antibacterial activity against drug-resistant bacteria both in vitro and in vivo, with the killing efficiency up to 99.99% for ampicillin-resistant Escherichia coli (AmprE. coli) and 99.9999% for methicillin-resistant Staphylococcus aureus (MRSA), indicating the exceptional bactericidal activity of the newly developed nanobiocide. Scheme 1 | Multimodal and synergistic killing of drug-resistant bacteria. (a) Schematic fabrication of Poly-Cu nanoparticles. (b) The mechanism of action of Poly-Cu nanoparticles on the multimodal and synergistic killing of drug-resistant bacteria, in which ɛ and η represent the molar extinction coefficient and photothermal conversion efficiency, respectively. Download figure Download PowerPoint Experimental Methods Fabrication of nanoparticles made of cupric acetate-doped LA via in situ surface polymerization To facilitate in situ surface polymerization, the double-bond-modified LA (1.0 wt %; see Supporting Information for chemical structure and synthetic route) was first mixed into the greenish-blue solids of cupric acetate/LA mixture (weight ratios of cupric acetate = 0%, 3.0%, 5.0%, and 8.0%, respectively). The mixture was then dissolved in tetrahydrofuran (THF) at a final concentration of 100 mg mL−1. The neutral monomer acrylamide (AAM) and positively charged monomer N-(3-aminopropyl)methacrylamide hydrochloride (APM) were prepared as aqueous stock solutions at concentrations of 200 and 100 mg mL−1, respectively. The stock solution of the cross-linker N,N’-methylene bisacrylamide (MBA) was prepared in anhydrous dimethyl sulfoxide (DMSO) at a concentration of 100 mg mL−1. The aforementioned stock solutions were mixed together at the molar ratio of the greenish-blue solids, AAM, APM, and MBA in 1:30,000:1000:1500 (300 μL), followed by dropwise addition into water (700 μL). After introducing tetramethylethylenediamine (TEMED), the mixture was subjected to ultrasonication for 2 min. Next, radical polymerization was initiated on the surface of the resultant nanoparticles by adding initiator ammonium persulfate (APS; the molar ratio of greenish-blue solids/APS/TEMED = 1:1000:2000). The polymerization was allowed to proceed at 0 °C for 6–8 h; then, the solution was dialyzed against water overnight to remove water-soluble byproducts and unreacted monomers. The generated copper-containing nanoparticles were denoted as Poly-Cu nanoparticles. Besides, the nanoparticles composed of pure LA were also prepared using an identical method (denoted as Poly-LA nanoparticles), in which the greenish-blue solids of cupric acetate/LA mixture were replaced by pure LA. To visualize the interactions between the nanoparticles and bacteria, an aggregation-induced emission luminogen (AIEgen; see Supporting Information for chemical structure and synthetic route) was introduced into the nanoparticles following an identical procedure, except for the substitution of the greenish-blue solids with LA and AIEgen mixture (weight ratio, LA/AIEgen = 99∶1). The resultant nanoparticles were denoted as Poly-AIE nanoparticles. All nanoparticle suspensions were filtered through 0.8-μm membranes prior to use. Bacterial culture The AmprE. coli and MRSA were provided by Prof. Qiong Yang (College of Life Sciences, Beijing Normal University, Beijing, China) and Prof. Jianfeng Liu (Institute of Radiation Medicine, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin, China), respectively. For the AmprE. coli culture, a single colony of AmprE. coli (Gram-negative) on a solid Luria–Bertani (LB) agar plate was transferred to 5 mL LB broth medium with 100 μg mL−1 ampicillin sodium in a shaking incubator (170 rpm) at 37 °C overnight. The bacterial cells were harvested by centrifugation (3500 g for 5 min) and washed three times with phosphate-buffered saline (PBS; 10 mM, pH = 7.4). The supernatant was discarded, and the remaining AmprE. coli was resuspended in PBS, which was diluted to an optical density of 1.5 at 600 nm (OD600 = 1.5) for subsequent use. For the culture of MRSA, a single colony of MRSA (Gram-positive) on a solid Tryptic Soy Broth (TSB) agar plate was transferred to 5 mL of TSB broth medium with 100 μg mL−1 tetracycline hydrochloride in a shaking incubator (170 rpm) at 37 °C overnight. The bacterial cells were harvested by centrifugation (3500 g for 5 min) and washed three times with PBS. The supernatant was discarded, and the remaining MRSA was resuspended in PBS, which was diluted to an optical density of 1.0 at 600 nm (OD600 = 1.0) for subsequent use. In vitro antibacterial experiments To study the antibacterial effect of Poly-Cu nanoparticles in vitro, six groups of experiments were tested without and with NIR irradiation, using both AmprE. coli and MRSA in parallel. The experimental setup included the following: the control groups treated with PBS only [denoted as (1) Blank and (2) Blank + NIR], the experimental groups treated with Poly-LA nanoparticles [5 mg mL−1; denoted as (3) Poly-LA and (4) Poly-LA + NIR], and the experimental groups treated with Poly-Cu nanoparticles [5 mg mL−1; denoted as (5) Poly-Cu and (6) Poly-Cu + NIR]. The bacteria (AmprE. coli, OD600 = 0.75; MRSA, OD600 = 0.50) were incubated for 1 and 5 h at 37 °C on a shaking incubator (170 rpm). In terms of the groups with NIR laser irradiation, the bacteria were first incubated for 10 min, followed by 808-nm laser irradiation at a power density of 1.5 W cm−2 for 10 min. Once the incubation was completed, the bacterial suspensions were diluted by 5 × 104 fold with PBS. 100 μL of the bacterial solution was then used for plate counting, in which AmprE. coli cell suspension was spread on LB plates with 100 μg mL−1 ampicillin sodium and MRSA cell suspension was spread on TSB plates with 100 μg mL−1 tetracycline hydrochloride. Then the plates were cultured at 37 °C for 15–18 h. The colony-forming units (CFU) number of each plate was counted, which was used for quantitative antibacterial evaluation (n = 3 for each group). In vivo antibacterial experiments All animal procedures were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals of Peking Union Medical College, and all animal experiments were approved by the Animal Experiments and Ethics Review Committee of the Institute of Radiation Medicine, Chinese Academy of Medical Sciences. Specifically, female BALB/c mice (6–8 weeks old) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China) and raised in our specific-pathogen-free (SPF) grade laboratory. Prior to the creation of skin injury, the mice were intraperitoneally injected with cyclophosphamide (80 mg kg−1) to compromise their immunity against the inoculated bacteria. At 24-h postinjection, a round wound with a diameter of 1 cm was created in the right back of the mouse using a skin punch, followed by wound infection with 100 μL of AmprE. coli (OD600 = 0.5) or MRSA (OD600 = 0.2). The wound of each mouse was then bounded with gauze, and the day of the bacterial inoculation was defined as the zero-day. At 24-h postinoculation, the infected mice were randomly divided into six groups (n = 3), and 100 μL of each cell suspension was added onto the wound area, including the control groups treated with PBS (denoted as Blank and Blank + NIR), the experimental groups treated with Poly-LA nanoparticles (5 mg mL−1, denoted as Poly-LA and Poly-LA + NIR), and the experimental groups treated with Poly-Cu nanoparticles (5 mg mL−1, denoted as Poly-Cu and Poly-Cu + NIR). All NIR groups were irradiated under an 808-nm laser at a power density of 1.5 W cm−2 for 5 min and the temperature was dynamically monitored using the FLIR E6 infrared camera (FLIR Systems, Inc., Wilsonville, USA). Additionally, the healing process of the infected areas and body weights of the infected mice were recorded by a digital camera and balance throughout the therapeutic window (10 days), in which the damaged areas were measured and quantified by the ImageJ software (National Institutes of Health, Bethesda, MD). To assess the in vivo therapeutic effect and biosafety issue, the mice were sacrificed at the end of the treatment, and half of the infected skin tissue as well as the major organs (including heart, liver, spleen, lung, and kidney) were collected and fixed in 4% paraformaldehyde for 12 h. These tissues were subjected to hematoxylin and eosin (H&E) staining, then examined under a digital microscope. To determine the number of bacteria at the infection site, the other half of the infected skin tissue was homogenized in PBS (500 μL), and the diluted tissue solutions (10 times) were used for plate counting after incubation at 37 °C for 20 h (n = 3). Statistical analysis The results were presented as mean ± standard deviation, with “n” indicating the number of samples per group. Significant differences between groups were analyzed using one-way analysis of variance (ANOVA) with Tukey’s post hoc test (*p < 0.05; **p < 0.01; ***p < 0.001). Results and Discussion Turning cupric acetate into efficient photothermal materials with LA We started this study by demonstrating the chelation capability of LA in toluene toward copper ions in aqueous cupric acetate (Figure 1a). In a demonstration experiment, aqueous cupric acetate was added to a glass vial, followed by introducing a toluene solution of LA (Phases 5 and 6). The glass vial was subjected to liquid–liquid extraction by vigorous shaking. After standing for a while for complete phase separation, we observed that LA dissolved in toluene could extract copper ions efficiently from the aqueous phase to the organic phase and form a greenish-blue complex (arising from d–d transitions; Phases 7 and 8). To verify that the color change occurred only in the presence of LA, a control experiment was performed by substituting the toluene solution of LA with pure toluene (Phases 1 and 2). In this case, however, vigorous shaking did not cause any remarkable color change (Phases 3 and 4), indicating the pivotal role of LA during copper extraction. To quantify such a process, the UV–vis–NIR spectra of all solutions corresponding to Phases 1–8 were measured (Figure 1b). Based on the absorption values before and after shaking, the extraction efficiencies of copper ions from its aqueous solution to toluene in the absence and presence of LA were calculated to 2% and 81%, respectively. Besides, the molar extinction coefficient (ɛ) of the greenish-blue complex exhibited a 12-fold enhancement relative to that of the aqueous cupric acetate (18 M−1 cm−1). We speculated that the remarkable changes in both color and absorbance were attributed to the strong chelation capability of LA toward copper ions, which was achieved by donating a proton to an acetate ion to form acetic acid that was highly miscible with water. In the toluene phase, the dimeric complex was formed with the formula of [Cu(C11H23COO)2·H2O]2 or [Cu(C11H23COO)2·C11H23COOH]2 as indicated in a previous report,45 in which each copper ion was coordinated by four monodentate laurate that bridged two copper ions in a paddlewheel structure (see the inset in Figure 1b), similar to the crystal structures of Cu(CH3COO)2·H2O or anhydrous Cu(CH3COO)2.46 As water molecules that served as the ligand of copper ions in the aqueous phase were displaced by the laurate in toluene, the new hybridization of d electrons in copper ions reduced the orbital symmetry, which improved the probability of d–d transitions, and thus, resulted in an enhanced absorption in the NIR region.47 It should be noted that, compared to the aqueous cupric acetate (Phase 6), the resultant extract in toluene (Phase 7) exhibited a relatively blue-shifted UV–vis–NIR spectrum. We inferred that the laurate ions are a class of stronger-field ligands than acetate ions, and the larger crystal-field splitting energy between eg and t2g states shifted the absorption peak to a shorter wavelength.47 To further confirm that the ligand exchange was promoted by the donation of a proton from LA, the pH values of all aqueous phases were tested (Figure 1c). Interestingly, after extraction by LA, the aqueous solution exhibited a decreased pH (4.19 ± 0.01) relative to the one before extraction (5.75 ± 0.02), as well as the one extracted by pure toluene (5.78 ± 0.04). This result indicated that the process of copper chelation that occurred at the water–toluene interface was achieved by transferring a proton from LA to the aqueous phase. Figure 1 | Turning cupric acetate and LA into efficient photothermal materials. (a) A digital photograph showing the extraction of copper ions in aqueous cupric acetate to toluene in the absence and presence of LA before (S−) and after (S+) vigorous shaking. (b) UV–vis–NIR spectra of the samples corresponding to Phases 1–8 as denoted in (a). The inset indicates the chemical structure of the paddlewheel dimer of the copper–LA complex. (c) A table showing the pH values of Phases 2/6, 4, and 8. (d) Changes in the temperature of LA doped with different weight ratios of cupric acetate under an 808-nm laser at a power density of 0.5 W cm−2. (e) Photothermal heating behavior of LA doped with 1.5 wt % cupric acetate (LA + 1.5% Cu) after repeated cycles of laser irradiation. (f) DSC curves of LA and LA + 1.5% Cu solids. Download figure Download PowerPoint Considering the remarkably enhanced absorption in the NIR region, we further studied the photothermal property of the greenish-blue complex. First, a series of LA solids with varying weight ratios of cupric acetate were fabricated by heating the mixture to a temperature higher than the melting point of LA. It should be noted that during the dissolution of cupric acetate in LA, an acidic odor that smelled like acetic acid was identified, indicating the occurrence of ligand exchange by releasing acetic acid molecules. Next, the resultant greenish-blue solids were irradiated by an 808-nm laser, the entire process of which was recorded using an infrared camera in a real-time fashion. As shown in Figure 1d, all the greenish-blue solids exhibited a concentration-dependent enhancement in heat generation, with the temperatures rising up to 54, 68, 76, and 78 °C, respectively, which was remarkably different from pure LA that had no photothermal effect. In view of the similar photothermal behavior, LA doped with 1.5 wt % cupric acetate was selected for subsequent photothermal characterization. We further assessed the photothermal stability of the greenish-blue solid during repeated laser irradiation (Figure 1e). Even after five cycles of laser irradiation, no obvious decrease in the photothermal performance was found, indicating its superior photostability over conventional organic photothermal dyes (e.g., indocyanine green19). To examine the impact of cupric acetate on the thermal property of LA, we measured the differential scanning calorimetry (DSC) curves of pure LA and LA doped with 1.5 wt % cupric acetate, with the phase-transition temperatures of 44.81 and 44.67 °C, respectively (Figure 1f). Considering the negligible temperature variation, it is confirmed that LA doped with 1.5 wt % cupric acetate had similar thermal behavior to pure LA. Formulation of cupric acetate-doped LA into nanoparticles via in situ surface polymerization Next, we intended to formulate these photothermal materials into nanoparticles for antibacterial studies. Since transforming bulk materials into nanoparticles typically results in decreased photothermal performance due to solvent dilution, we increased the doping ratio of cupric acetate to 5.0 wt % in LA. The solid-state UV–vis–NIR spectrum of LA doped with 5.0 wt % showed essentially identical absorption to cupric acetate but enhanced absorption at the same concentration of copper ( Supporting Information Figure S1). Besides, the DSC curve of LA doped with 5.0 wt % cupric acetate did not exhibit a marked change relative to pure LA, with the phase-transition temperature of 44.42 °C ( Supporting Information Figure S2). To apply this photothermal material to biomedical use, determining the binding affinity between copper ions and LA would be critical, as the release of large quantities of copper ions could result in adverse effects. It is well-known that ethylenediaminetetraacetic acid disodium salt (EDTA) has a strong capacity to chelate copper ions by forming a stable Cu–EDTA complex. As such, an EDTA challenge test was performed by incubating the cupric acetate-doped LA both in solid and melted states with 50–500-fold excess of EDTA ( Supporting Information Figure S3). The UV–vis–NIR spectra data demonstrated that the copper ions were barely extracted from the solid complex ( Supporting Information Figure S3a), while 50-, 100-, and 500-fold excess of EDTA resulted in 6.2%, 9.4%, and 15.6% extraction of copper ions from the melted complex, indicating the strong coordination capability of LA with copper ions ( Supporting Information Figure S3b). The cupric acetate-doped LA was then formulated into colloidally dispersed nanoparticles via a technique that combined nanoprecipitation and in situ surface polymerization. Specifically, the cupric acetate-doped LA was mixed with 1.0 wt % double-bond-modified LA (see Supporting Information for chemical structure and synthetic route), followed by dissolution in THF and dropwise addition into an aqueous solution under vigorous vortexing. The neutral monomer AAM, positively charged monomer APM, cross-linker MBA, and catalyst TEMED were added to the solution to initiate in situ radical polymerization under the participation of APS at 0 °C. After dialysis against water, the copper-containing LA nanoparticles were formed (denoted as Poly-Cu nanoparticles). As a control, we also fabricated nanoparticles without the introduction of cupric acetate into LA using the identical method (denoted as Poly-LA nanoparticles). Transmission electron microscopy (TEM) was used to characterize the morphology of the resultant nanoparticles. As shown in Figure 2a and Supporting Information Figure S4a, both Poly-Cu and Poly-LA nanoparticles had a spherical morphology. Dynamic light scattering (DLS) results showed that the hydrodynamic diameters ranged from 70 to 300 nm, with average diameters of 127 and 120 nm for Poly-Cu and Poly-LA nanoparticles, respectively (Figure 2b and Supporting Information