\documentclass[10pt]{article}

\usepackage{fullpage}
\usepackage{setspace}
\usepackage{parskip}
\usepackage{titlesec}
\usepackage[section]{placeins}
\usepackage{xcolor}
\usepackage{breakcites}
\usepackage{lineno}
\usepackage{hyphenat}





\PassOptionsToPackage{hyphens}{url}
\usepackage[colorlinks = true,
            linkcolor = blue,
            urlcolor  = blue,
            citecolor = blue,
            anchorcolor = blue]{hyperref}
\usepackage{etoolbox}
\makeatletter
\patchcmd\@combinedblfloats{\box\@outputbox}{\unvbox\@outputbox}{}{%
  \errmessage{\noexpand\@combinedblfloats could not be patched}%
}%
\makeatother


\usepackage{natbib}




\renewenvironment{abstract}
  {{\bfseries\noindent{\abstractname}\par\nobreak}\footnotesize}
  {\bigskip}

\titlespacing{\section}{0pt}{*3}{*1}
\titlespacing{\subsection}{0pt}{*2}{*0.5}
\titlespacing{\subsubsection}{0pt}{*1.5}{0pt}


\usepackage{authblk}


\usepackage{graphicx}
\usepackage[space]{grffile}
\usepackage{latexsym}
\usepackage{textcomp}
\usepackage{longtable}
\usepackage{tabulary}
\usepackage{booktabs,array,multirow}
\usepackage{amsfonts,amsmath,amssymb}
\providecommand\citet{\cite}
\providecommand\citep{\cite}
\providecommand\citealt{\cite}
% You can conditionalize code for latexml or normal latex using this.
\newif\iflatexml\latexmlfalse
\providecommand{\tightlist}{\setlength{\itemsep}{0pt}\setlength{\parskip}{0pt}}%

\AtBeginDocument{\DeclareGraphicsExtensions{.pdf,.PDF,.eps,.EPS,.png,.PNG,.tif,.TIF,.jpg,.JPG,.jpeg,.JPEG}}

\usepackage[utf8]{inputenc}
\usepackage[ngerman,greek,english]{babel}



\usepackage{float}








\begin{document}

\title{Nano-antibiotics combined with laser irradiation to reduce the
development of drug resistance of K. Pneumoniae and accelerate wound
healing}



\author[1]{Tiansheng Liu}%
\author[2]{Guowei Zhong}%
\author[2]{Dongying Tang}%
\author[3]{Xu Liu}%
\author[1]{Xianghua Zhong}%
\author[1]{Yuejun Yang}%
\author[2]{Chunyi Tong}%
\author[1]{Xiaoping Yang}%
\affil[1]{Hunan Normal University}%
\affil[2]{Hunan University}%
\affil[3]{Hunan University of Chinese Medicine}%


\vspace{-1em}



  
  \date{June 17, 2024}


\begingroup
\let\center\flushleft
\let\endcenter\endflushleft
\maketitle
\endgroup





\selectlanguage{english}
\begin{abstract}
Bacterial infections, especially infections caused by multi-drug
resistant bacteria, pose a serious threat to human health and bring huge
challenges to clinical treatment. The excessive use of antibiotics can
easily lead to the emergence of bacterial resistance, which severely
limits clinical treatment options. There is an urgent need to develop
high-efficiency antibacterial materials and treatment strategies to
inhibit infections caused by multidrug-resistant bacteria. In this work,
a nanocomposite named Ofloxacin@HMPB@HA(OHH NPs) combined with the laser
irradiation was used to reduce the development of drug resistance and
accelerate wound healing in a model infected by Klebsiella
pneumoniae(K.Pneumoniae). In vitro results showed that compared with OHH
NPs or NIR laser irradiation alone, this combination strategy can exert
a synergistic effect on anti-K.Pneumoniae by destroying cell integrity
with generating ROS and reducing ATP, and also inhibit the development
of bacterial resistance. Moreover, in vivo experiments have shown that
the system effectively promotes wound healing through killing
K.Pneumoniae and promoting the formation of new tissues. In summary,
these results indicate that OHH NPs show great potential in the clinical
application of bacterial infections.%
\end{abstract}%



\sloppy


\textbf{Nano-antibiotics combined with laser irradiation to reduce the
development of drug resistance of K. Pneumoniae and accelerate wound
healing}

Tiansheng Liu\textsuperscript{1\#}, Guowei Zhong\textsuperscript{2\#},
Dongying Tang\textsuperscript{2}, Xu Liu\textsuperscript{3}, Jialong
Fan\textsuperscript{2}, Xianghua Zhong\textsuperscript{1}, Yuejun
Yang\textsuperscript{1}*, Chunyi Tong\textsuperscript{2}*, Xiaoping
Yang\textsuperscript{1}

1 School of Medicine, Key Laboratory of Protein Chemistry and
Developmental Biology of Fish of Ministry of Education, Hunan Normal
University, Changsha, 410125, PR China.

2 College of Biology, Hunan University, Changsha, 410082, PR China

3 School of Pharmacy, Hunan University of Chinese Medicine, Changsha
410208, China

\# These authors contributed to the work equally and should be regarded
as co-first authors.

* To whom correspondence should be addressed.

\textbf{Abstract}

Bacterial infections, especially infections caused by multi-drug
resistant bacteria, pose a serious threat to human health and bring huge
challenges to clinical treatment. The excessive use of antibiotics can
easily lead to the emergence of bacterial resistance, which severely
limits clinical treatment options. There is an urgent need to develop
high-efficiency antibacterial materials and treatment strategies to
inhibit infections caused by multidrug-resistant bacteria. In this work,
a nanocomposite named Ofloxacin@HMPB@HA(OHH NPs) combined with the laser
irradiation was used to reduce the development of drug resistance and
accelerate wound healing in a model infected by \emph{Klebsiella
pneumoniae(K.Pneumoniae)} . In vitro results showed that compared with
OHH NPs or NIR laser irradiation alone, this combination strategy can
exert a synergistic effect on anti-\emph{K.Pneumoniae} by destroying
cell integrity with generating ROS and reducing ATP, and also inhibit
the development of bacterial resistance. Moreover, in vivo experiments
have shown that the system effectively promotes wound healing through
killing K.Pneumoniae and promoting the formation of new tissues. In
summary, these results indicate that OHH NPs show great potential in the
clinical application of bacterial infections.

\textbf{Keywords}

Hollow Mesoporous Prussian Blue, antibiotic resistance, antibacterial
nanoplatform, photothermal therapy

\textbf{Introduction}

Over the past decades, the ever-growing emergence of bacterial
infections causes devastating consequences to public health
worldwide\textsuperscript{1}. Clinically, the most widely employed and
effective therapeutic method of bacterial infections is antibiotics
treatment\textsuperscript{2-3}. However, the overuse of antibiotics has
given rise to the appearance of multidrug-resistant (MDR) bacteria which
weakens the effectiveness of most antibiotics\textsuperscript{4}.
Moreover, high-dose antibiotics treatment has great potential to induce
severe adverse effects and systemic toxicity such as angioedema,
thrombophlebitis, anaphylactic shock and so on\textsuperscript{5-6}.
Thus, there is an urgent requirement for development of new effective
antibacterial strategies which make rational use of conventional
antibiotics and minimize antibiotic dose.

Recently, nanoparticle (NP)-based local ``on-demand'' antibacterial
drugs delivery systems have received burgeoning attention since they can
prolong drugs retention at the infected area with low undesired drugs
diffusion, improved therapeutic efficacy and reduced
toxicity\textsuperscript{7-9}. For instance, Gu and co-workers reported
levofloxacin hydrochloride-loaded, silver core-embedded mesoporous
silica for synergistically combating drug-resistant bacterial
infections\textsuperscript{10}; Wu and co-workers developed
ciprofloxacin-loaded photothermal PDA/GC hydrogel for combined
chemo-photothermal therapy of bacterial infections\textsuperscript{11};
Han and co-workers successfully fabricated rifampicin-loaded endogenous
stimulus--responsive liposome nanoreactors for a combined therapy of
bacterial infections\textsuperscript{12}. Although NP-based drugs
delivery systems can effectively enhance antibacterial efficacy and
lower biological toxicity of antibacterial drugs, therapeutic effect of
single modality of chemotherapy based on drugs delivery systems is still
unsatisfactory due to poor diffusivity of drugs in targeted
sites\textsuperscript{13}, which will seriously hamper their practical
applications in clinic. Chemotherapy-based synergistic therapy is a
particularly encouraging strategy to strengthen the bactericidal effect
for combating bacterial infections\textsuperscript{14} .

Photothermal therapy (PTT) triggered by near-infrared (NIR) laser has
been applied as one of the most effective antibacterial
strategies\textsuperscript{15-16}. PTT can cause bacterial death through
hyperthermia-induced denaturation of bacterial enzymes and irreversible
bacterial destruction\textsuperscript{17}. Comparing with
antimicrobials, this strategy presented many advantages for
antibacterial application as follow: (1) NIR laser has high spatial
resolution and a deep penetration ability to tissues with minimal
invasiveness\textsuperscript{18}; (2) PTT can efficiently kill bacteria
in a short time (only a few minutes)\textsuperscript{19-20}; (3) PTT has
broad-spectrum antibacterial ability and can efficiently eliminate MDR
bacterial strains with less probability to induce drug
resistance\textsuperscript{21}; (4) NIR laser can focus on a targeted
area to accelerate blood circulation with little influence on the whole
body\textsuperscript{22-23}. Unfortunately, the locally high temperature
required to completely eradicate the bacteria may cause severe thermal
injury to the surrounding healthy tissues\textsuperscript{24}.
Combination of chemotherapy and PTT is an especially promising approach
for overcoming the limitations of PTT alone.

To date, numerous nanomaterials have been developed as photothermal
agents, including palladium NPs\textsuperscript{25}, carbon
nanomaterials\textsuperscript{26}, black phosphorus\textsuperscript{27},
Au nanorods\textsuperscript{28} and so on. Among these nanomaterials,
Prussian blue (PB) approved by USA Food and Drug Administration (FDA) as
a clinical drug gains great popularity for PTT due to the eminent
advantages of strong NIR radiation absorption ability, high photothermal
efficiency, excellent chemical stability, splendid biocompatibility and
low-cost\textsuperscript{29-30}. More encouragingly, we have
successfully prepared hollow mesoporous PB (HMPB) that has larger drug
loading capacity owing to interior hollow cavity and more rough
mesoporous surface compared with PB. Simultaneously, HMPB possess almost
the same photothermal ability as PB. These unique features of HMPB make
it an ideal photothermal agent as well as drug carrier, which can
effectively combine chemotherapy and PTT\textsuperscript{31}.

Herein, we fabricated a robust surface-adaptive, on-demand antibacterial
nanoplatform by covering hyaluronic acid (HA) on ofloxacin loaded HMPB
to exert the strong synergistic antibacterial effect involving
antibiotic and PTT killing. As showed in Scheme 1, for the first time,
we utilized the hyaluronic acid (HA)-modified HMPB as a nanocarrier to
load the ofloxacin, a traditional antibiotic with broad spectrum of
antibacterial property against both gram-positive and gram-negative
bacteria, even MDR bacteria. HA with excellent biocompatibility was
employed as the capping agent to prevent unwanted ofloxacin release in
this system. Meanwhile, HA on the surface of HMPB acted in the role of
``on-demand'' releasing agent which can be degraded by hyaluronidase
(HAase) secreted in numerous bacteria. The encapsulated ofloxacin could
be released and further efficiently combat pathogenic bacteria at the
infected area when the coating layer HA of the resulted OHH NPs was
degraded by overexpressed HAase generated by bacteria. The intelligent
behavior of ``on-demand'' release of this nanoplatform could
considerably enhanced bioavailability of ofloxacin and reduced toxicity
of ofloxacin. Additionally, under NIR light irradiation, the nanocarrier
showed photothermal effect, which could generate locally increased
temperature to inactivate bacteria. In summary, the OHH NPs exhibited
surface-adaptive characteristic and on-demand synergistically
antibacterial capability originated from the released ofloxacin and
produced hyperthermia under NIR light irradiation, which could
significantly improve the antibacterial efficacy and lowered the dosage
of ofloxacin. Furthermore, this powerful antibacterial nanoplatform was
successfully employed for the treatment of \emph{K.pneumoniae} -infected
wound.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

\textbf{2.} \textbf{Materials and Methods}

\textbf{2.1. Materials}

K\textsubscript{3}{[}Fe(CN)\textsubscript{6}{]}*3H\textsubscript{2}O was
brought from Fuchen Chemical Reagent Factory (China). Poly
(N-vinylpyrrolidone) (PVP) was obtained from Zhanyun Chemical Co., Ltd.
(China). Ofloxacin, Haluronic acid and Polyacrylic acid were obtained
from Macklin Biotechnology Co., Ltd (China).
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and
N-hydroxysuccinimide (NHS) were purchased from Sigma Aldrich. All other
chemicals and solvents were of reagent grade.

\textbf{2.2. Preparation of PBNPs.}

PB NPs were synthesized according to the previous method developed by
our group. In brief, 264 mg
K\textsubscript{3}{[}Fe(CN)\textsubscript{6}{]}*3H\textsubscript{2}O and
3 g PVP were dissolved in 40 mL of 0.01 M HCl solution under stirring
for 1 h at room temperature. Then, the mixture solution was heated at 80
\selectlanguage{ngerman}°C for 20 h with oil bath. After centrifugation and water washing for 3
times, the precipitate was dried under vacuum for the preparation of PB
NPs.

\textbf{2.3} \textbf{Preparation of HMPB NPs.}

HMPB NPs were prepared following the previous method with little
modification\textsuperscript{32}. 100 mg PVP and 20 mg PB NPs were
dissolved in 20 mL of 1 M HCl solution under magnetic stirring for 3 h.
Then, the mixture solution was transferred into a stainless autoclave
and heated at 140 °C for 4 h. After acid etching, HMPB NPs is collected
by centrifugation and washed three times with deionized water.

\textbf{2.4. Preparation of OHH NPs.}

For synthesis of OHH NPs, 2 mg HMPB NPs and 2 mg ofloxacin were added to
1 mL of deionized water with stirring for 4 h. Then remove unbound
ofloxacin by centrifugation. After centrifugation, 6 mg polyacrylic acid
(PAA) was added into the mixture. Furthermore, the above solution was
added 6 mg of EDC and 6 mg of NHS. After stirring for 2 h, 3 mg
Haluronic acid (HA) was added into the mixed solution and further
reacted overnight. Finally, OHH NPs were extracted by centrifugation.

\textbf{2.5. Characterization of hybrids}

SEM mapping, FTIR, X-ray diffraction (XRD) and X-ray photoelectron
spectroscopy (XPS) were tested in Guangdong Puchuan Inspection Co, Ltd.,
China. UV--Vis absorption spectra were recorded with a UV-1800
spectrophotometer (Shimadzu, Japan). The hydrodynamic diameters and zeta
potential of NPs were measured using a Zeta-sizer Nano ZS (Malvern, UK).
The morphology of NPs was characterized by transmission electron
microscope (TEM, JEM-2100F, JEOL).

\textbf{2.6. Photothermal effect ofOHH NPs}

0.6 mL of PBS, HMPB NPs, ofloxacin or OHH NPs solution in Eppendorf tube
was exposed to 808 nm NIR laser with a laser density of 1
W/cm\textsuperscript{2} for 10 min. An infrared thermal camera was used
to capture the infrared thermal images and record the real-time
temperature every 20s. To assess the photostability of OHH NPs, the
real-time temperature of OHH NPs (100 \selectlanguage{greek}μ\selectlanguage{english}g/mL) solution was recorded in
five cycles with 5 min laser on and 10 min laser off.

\textbf{2.7. In vitro antibacterial experiments}

Methicillin-resistant \emph{Staphylococcus aureus} (MRSA)
and\emph{K.pneumoniae} were cultivated separately overnight at 37 \selectlanguage{ngerman}°C in
Luria--Bertani broth (LB broth). The antibacterial effect of the
nanomaterials was determined by the agar diffusion method. Briefly, 100
\selectlanguage{greek}μ\selectlanguage{english}L bacterial suspension (10\textsuperscript{8} CFU
mL\textsuperscript{-1}) of MRSA or \emph{K.pneumoniae} was spread
homogeneously on the agar plate. Then, 25 \selectlanguage{greek}μ\selectlanguage{english}L of sample (PBS, HMPB NPs,
ofloxacin and OHH NPs) dropped to a filter paper on MRSA
and\emph{K.pneumoniae} -coated agar plates. The agar plates were
transferred into the 37 \selectlanguage{ngerman}°C incubator. After incubation for 12 h, the
diameters of inhibition area were measured to assess the bactericidal
activity.

The plate counting method was adopted to test \emph{in
vitro}antibacterial efficiency of sample (PBS, HMPB NPs, ofloxacin and
OHH NPs) combined with NIR laser irradiation. 200 \selectlanguage{greek}μ\selectlanguage{english}L of bacterial
suspension (1\selectlanguage{ngerman}×10\textsuperscript{4} CFU mL\textsuperscript{-1}) was
incubated with PBS (control), HMPB, ofloxacin or OHH NPs and then
irradiated with/without 808 nm laser at a power density of 1
W/cm\textsuperscript{2} for 5 min. Afterwards, 100 \selectlanguage{greek}μ\selectlanguage{english}L of appropriately
diluted bacterial suspension was spread on LB agar plate followed by
incubated at 37\selectlanguage{ngerman}°C for 24 h. Bacterial colony forming units were counted
and antibacterial efficiency was calculated according to the following
equation:

Survival ratio (\%) =
CFU\textsubscript{sample}/CFU\textsubscript{control}×100\%

\textbf{2.8. Live/dead staining and ROS detection assays}

The bacterial viability was investigated using Live/Dead Bacterial
Viability Kit. \emph{K.pneumoniae} after different treatments was
collected by centrifuging (8000 rpm, 5 min) and then co-dyed with
Calcein-AM and propidium iodide (PI) in dark for 30 min. After that, the
bacteria were washed with normal saline to remove excess dye. Finally,
bacterial solutions were dropped on the glass slides and imaged Confocal
Microscope (FV1200, Olympus).

For the analysis of intracellular ROS content, 10\textsuperscript{8} CFU
mL\textsuperscript{-1} \emph{K.pneumoniae} cells were added into the PBS
(control), HMPB (100 \selectlanguage{greek}μ\selectlanguage{english}g/mL), ofloxacin (10 \selectlanguage{greek}μ\selectlanguage{english}g/mL), and OHH NPs (100
\selectlanguage{greek}μ\selectlanguage{english}g/mL) and further incubated for 30 min, the net concentration of the
HMPB substrate in all sample groups has been maintained at 100 \selectlanguage{greek}μ\selectlanguage{english}g/mL.
After that, the \emph{K.pneumoniae} cells were rinsed with medium and
stained with DCFH-DA under 37 \selectlanguage{ngerman}°C for 20 min and then analyzed using a
confocal microscope (FV1200, Olympus). Quantitative determination of the
ROS content in the cell samples was further carried out as a complement
to the characterizations above.

\textbf{2.9. Morphological study of bacteria}

The \emph{K.pneumoniae} cells treated by different samples were
collected after centrifugation (8000 rpm, 5 min) and rinsed three times
with normal saline. Thereafter, the treated bacteria were fixed with
2.5\% glutaraldehyde solution in dark for 4 h at 4 °C. Subsequently, the
fixed samples were dehydrated for 10 min with sequential concentrations
(30\%, 50\%, 70\%, 80\%, 90\%, 95\% and 100\%) of ethanol solution,
respectively, followed by observing under a scanning electron
microscope.

\textbf{2.10. ATP assay}

The metabolic activity of bacteria was assessed by an Enhanced ATP Assay
Kit (Beyotime). \emph{K.pneumoniae} cells treated with different samples
was harvested by centrifuging (8000 rpm,5 min) at 4 °C and resuspended
in 100 \selectlanguage{greek}μ\selectlanguage{english}L of lysis buffer. Subsequently, all samples were transferred
into an ultrasonic cell disruption instrument at 30\% power (3 s on, 5 s
off) for 5 min in an ice bath. Finally, the harvested supernatant was
determined on a multi-mode detection platform in luminance mode.

\textbf{2.11. Protein leakage study}

Protein leakage detection of bacteria was performed referring to our
previous method. In brief, 10\textsuperscript{8} CFU
mL\textsuperscript{-1} \emph{K.pneumoniae} was firstly incubated with
PBS (control), HMPB (100 \selectlanguage{greek}μ\selectlanguage{english}g/mL), ofloxacin (10 \selectlanguage{greek}μ\selectlanguage{english}g/mL), and OHH NPs (100
\selectlanguage{greek}μ\selectlanguage{english}g/mL) for 12 h at 37 \selectlanguage{ngerman}°C. Hereafter, bacterial suspension
of\emph{K.pneumoniae} was centrifuged at 8000 rpm for 5 min at 4 °C and
the harvested supernatant was immediately transferred into a 96-well
plate. The concentration of protein was determined using a BCA Protein
Assay Kit (Cat\#PC0020, Solarbio).

\textbf{2.12. In vitro antibiofilm assay}

For establishment of biofilms, 5×10\textsuperscript{8} CFU
mL\textsuperscript{-1} of \emph{K.pneumoniae} cells were added to
96-well plate(100 \selectlanguage{greek}μ\selectlanguage{english}L per well)and incubated under stationary conditions
at 37 \selectlanguage{ngerman}°C for 1 day. After biofilm formation, the medium was discarded
and the plates were washed with PBS to remove the planktonic bacteria.
Then 200 \selectlanguage{greek}µ\selectlanguage{english}L of HMPB(100\selectlanguage{greek}μ\selectlanguage{english}g/mL), ofloxacin(10 \selectlanguage{greek}μ\selectlanguage{english}g/mL) and OHH NPs with HMPB
concentration of 100 \selectlanguage{greek}μ\selectlanguage{english}g/mL was added to the plates, respectively. For
control group, 200 \selectlanguage{greek}µ\selectlanguage{english}L of PBS without nanomaterials was used. All groups
were incubated at 37\selectlanguage{ngerman}°C for 12 h. For the NIR irradiated group, 808 nm
laser at power density of 1 W/cm\textsuperscript{2} was used to
illuminate sample for 5 min. The media in each well was then removed,
and the plates were washed carefully with PBS one time. The biofilm in
each well was fixed with methanol for 15 min and stained with 0.1\%
crystal violet for 15 min. After washing with Milli-Q water for three
times, ethanol (100 \selectlanguage{greek}µ\selectlanguage{english}L per well) was added to solubilize the crystal
violet staining. OD\textsubscript{560} in each well was read to
determine the biofilm formation.

Additionally, to obtain the 3D morphology of the
\emph{K.pneumoniae}biofilms, the biofilms formed under different
treatments was dyed by propidium iodide (PI) for 30 min in the dark.
After, the biofilms stained by PI were washed three times with PBS and
observed by a confocal laser scanning microscope (FV1200, Olympus). The
red fluorescence intensity of 3D images of biofilms was quantified using
Image J.

\textbf{2.13. Ofloxacin release behavior}

The measurement and calculation methods are based on our previous
reports\textsuperscript{33}. Briefly, 1 mL of OHH NPs solution with the
concentration of 100 \selectlanguage{greek}μ\selectlanguage{english}g/mL with/without HAase was irradiated by 808 nm
NIR laser at power density of 1 W/cm\textsuperscript{2} for 5 min. Then,
200 \selectlanguage{greek}μ\selectlanguage{english}L of solution was taken out every 30 min and the solution was
immediately centrifuged at 13,000 rpm for 10 min. Finally, the
concentration of ofloxacin of the supernatant was measured by UV-Vis
spectrophotometer. The drug loading capacity and entrapment efficiency
of the OHH NPs were calculated as follows:

Loading capacity (\%) = (M\textsubscript{T} -M\textsubscript{U})
/M\textsubscript{P} \selectlanguage{ngerman}×100 \%

where M\textsubscript{T} represents the total mass of drug,
M\textsubscript{U} represents the mass of the unencapsulated drug, and
M\textsubscript{P} represents the mass of PB and HMPB.

\textbf{2.14. In vitro cytotoxicity assay}

To investigate the \emph{in vitro} cytotoxicity of different
nanomaterials, NIH-3T3 and HUV-EC cells (1×10\textsuperscript{4}) were
seeded in a 96-well plate. After incubation for 24 h, the culture media
was removed and fresh culture media comprising different concentrations
of HMPB, ofloxacin or OHH NPs (0, 25, 50, 75, 100, 125 \selectlanguage{greek}μ\selectlanguage{english}g/mL). After 24
h incubation, the samples were washed with PBS three times and 100 \selectlanguage{greek}μ\selectlanguage{english}L of
CCK-8 solution was added into each well. After incubation for 2 h, cell
viability was measured by a microplate reader in the absorbance of 450
nm.

Hemolysis assay was performed using fresh Balb/c mouse blood. The fresh
mouse blood cells were harvested after centrifugation (3500 rpm, 5 min,
4 \selectlanguage{ngerman}°C). After that, 950 \selectlanguage{greek}μ\selectlanguage{english}L of PBS containing various concentrations of
nanomaterials are mixed with 50 \selectlanguage{greek}μ\selectlanguage{english}L of red blood cells. After incubation
for 2 h at 37 \selectlanguage{ngerman}°C, the samples were centrifuged (3500 rpm, 5 min, 4 °C)
and the optical density at 540 nm of the supernatant were determined on
a microplate reader. Each sample was measured in triplicate. PBS and
pure water were acted the role of a negative and positive control. The
ratio of hemolysis was calculated using the following equation:

Hemolysis (\%) = (A\textsubscript{sample} -
A\textsubscript{negative})/(A\textsubscript{positive} -
A\textsubscript{negative})×100\%;

Where A\textsubscript{sample} is the absorbance value of the supernatant
after the addition of HMPB, ofloxacin and OHH NPs, respectively.
A\textsubscript{negative} and A\textsubscript{positive} are the
absorbance values after the addition of PBS and pure water,
respectively.

\textbf{2.15. In vivo treatment of wound infection in Balb/c mice}

All procedures for animals were approved by the Institutional Animal
Care and Use Committee of Hunan University. Six-week-old female Balb/c
mice were obtained from Hunan SJA Laboratory Animal Co., Ltd. (Changsha,
China). \emph{K.Pneumoniae} was used as bacterial strain to infect mice.
All mice were randomly divided into five groups: PBS group, HMPB group,
ofloxacin group, OHH group and OHH+NIR group. A circular skin wound
(diameter of 10 mm) was prepared on the backbone of mice. Then, 100 \selectlanguage{greek}μ\selectlanguage{english}L
of \emph{K.pneumoniae} suspension (10\textsuperscript{8} CFU
mL\textsuperscript{-1}) was dropped on circular wounds for mouse
infection. Three days after continuous infection, 100 \selectlanguage{greek}μ\selectlanguage{english}L of PBS, HMPB,
ofloxacin OHH was smeared on the surface of the wounds every 2 days with
the same antibiotic concentration (10 \selectlanguage{greek}μ\selectlanguage{english}g mL\textsuperscript{-1}) and the
same HMPB concentration (100 \selectlanguage{greek}μ\selectlanguage{english}g mL\textsuperscript{-1}), respectively.
The OHH+NIR group was irradiated by 808 nm NIR laser (1
W/cm\textsuperscript{2}) for 5 min. Meanwhile, the diameter of the
wounds was measured and the wounds were photographed every 2 days.

On the 12th day, all mice were euthanized, and skin tissues and major
organs were excised to study their healing efficiency and biological
safety in vivo, respectively. In order to evaluate the antibacterial
effect in vivo, these infected tissues were also collected for coating
assay. Collect blood for measuring biochemical indicators.

\textbf{2.17. Statistical analysis}

Each data was expressed as mean \selectlanguage{ngerman}± standard deviation, and each
experiment is performed at least 3 times. Tukey's post-test was used for
one-way analysis of variance (ANOVA) for analysis. Among them,
*P\textless{}0.05, **P\textless{}0.01, ***P\textless{}0.001, the
difference between different groups is considered to be statistically
significant.

\textbf{3.Results and discussion}

\textbf{3.1. Preparation and characterization of OHH NPs}

The synthesis method of OHH NPs was schematically illustrated in Scheme
1. The morphology of the obtained nanomaterials was characterized
through transmission electron microscopy (TEM). The image of Fig. 1A
indicated that PB NPs was successfully prepared with a size of \selectlanguage{english}~100
nm\textsuperscript{32, 34-36}. HMPB NPs were synthesized on basis of PB
NPs by the controllable acid etching strategy, accompanying with
morphological change from square to spheric. Fig. 1B clearly showed
hollow mesoporous Prussian blue (HMPB) were successfully prepared with a
diameter of \selectlanguage{english}~75 nm and the hollow mesoporous structure. After loading
ofloxacin and coating with HA, the uniform size of OHH NPs slightly
increased to \selectlanguage{english}~78 nm (Fig. 1C), which was consistent with the result of
the dynamic light scattering (Fig. 1D). In Fig. 1E, the zeta potential
of HMPB increased from -19 mV to 8 mV after loading ofloxacin owing to
the positive charge of ofloxacin, while HA coating layer with a negative
charge caused the decrease of HMPB loaded ofloxacin from 8 mV to -15 mV
(Fig. 1E). The change of charge indirectly reflected the successful
synthesis of OHH NPs. The UV-vis absorption spectra of HMPB NPs,
ofloxacin and OHH NPs are shown in Fig. 1F. The as-obtained HMPB NPs
possessed a broad absorption in the NIR region with an absorption peak
at approximately 710 nm. After loading ofloxacin, a strong absorption
peak of OHH NPs appeared at 288 nm which was a characteristic absorption
of ofloxacin, indicating that ofloxacin was successfully loaded into
HMPB NPs. After coating with HA, the strong absorption peak of OHH NPs
red-shifted to 800 nm because of the absorbance of HA. Fig. 1G displayed
the FT-IR spectra of HMPB NPs, ofloxacin and OHH NPs. The characteristic
peak of HMPB NPs at 2086 cm\textsuperscript{-1} originated from the --CN
stretching vibration. Compared with HMPB NPs, new peaks of OHH NPs
appeared at 1414 cm\textsuperscript{-1}, 1708 cm\textsuperscript{-1} and
1630 cm\textsuperscript{-1}, which was assigned to the C=O, C=C and O-F
vibration of ofloxacin, respectively, further demonstrating the
successful loading of ofloxacin. Besides, X-ray photoelectron
spectroscopy (XPS) was also performed to investigate the chemical
composition of OHH NPs. As shown in Fig. 1H, the binding energy peak of
HMPB at 708.8 eV was attributed to Fe 2p. However, Fe 2p peak of OHH NPs
disappeared due to the presence of HA layer. In addition, obvious
binding energy peak located at 689.1 eV corresponding to F 1s was
observed from OHH NPs (Fig. S1), indicating successful loading of
ofloxacin. The X-ray diffraction (XRD) of OHH NPs showed the same
diffraction peak as that of HMPB (Fig. S2), indicating that the crystal
structure of HMPB loaded with ofloxacin has no effect. The as-above
characterization results solidly demonstrated that OHH NPs was
successfully prepared.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

\textbf{Fig. 1} . \textbf{Characterization of PB, HMPB and OHH NPs} .
(A-C)TEM images of PB, HMPB and OHH NPs. (D) Hydrodynamic sizes
distribution of PB, HMPB and OHH NPs determined by dynamic light
scattering (E) Zeta potentials of PB, HMPB and OHH NPs. (F) UV--Vis
spectra of NPs. (G,H) FT-IR spectra of NPs. (I) XPS survey scan of NPs.

\textbf{3.2. Photothermal effect of OHH NPs}

Due to the strong NIR absorption of OHH NPs, the photothermal effect of
OHH NPs under an 808 nm NIR light irradiation was systematically
assessed.\textsuperscript{34-35} As seen in Fig. 2A, the increased
temperature ([?]T) of HMPB NPs and OHH NPs solution was up to ~25 \selectlanguage{ngerman}°C after
irradiation with an 808 nm NIR laser at a power density of 1
W/cm\textsuperscript{2} for 10 min, whereas it was only \selectlanguage{english}~4.7 degC and \selectlanguage{english}~4.9
degC for pure water and ofloxacin, respectively. The negligible difference
of temperature increment between HMPB NPs and OHH NPs suggested that
ofloxacin loading and HA modification didn't influence the photothermal
property of HMPB NPs. Simultaneously, the real-time photothermal images
of Fig. 2B intuitively exhibited the discrepancy of photothermal ability
of different nanoparticals. Fig. 2C and Fig. 2D confirmed that the
temperature increase of OHH NPs could be precisely tuned by the
concentration of OHH NPs and laser intensity. Besides, the previously
reported equation\textsuperscript{37} was used to measure the
photothermal conversion efficiency (Fig. 2E). It could be calculated
that OHH NPs possessed a relatively high photothermal conversion
efficiency of 27.13\%. According to five heating-cooling cycles without
obvious temperature decrease, it could be concluded that OHH NPs
possessed the high photostability stability (Fig. 2F). All the
aforementioned results implied that OHH NPs could be utilized to be an
excellent photothermal agent for photothermal therapy(PTT).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
\end{figure}

\textbf{Fig. 2. Photothermal effect of OHH NPs} . (A) Photothermal
heating curve of water, HMPB, Ofloxacin and OHH NPs. (B) Infrared
thermal images of different samples (0.1 mg/mL) irradiated with 808 nm
light (1 W/cm\textsuperscript{2}, 10 minutes). (C) The heating curve of
OHH NPs under different power laser irradiation. (D) Temperature changes
of different concentrations of OHH solution irradiated by 808 nm laser.
(E) Photothermal effect of OHH NPs under 808 nm laser-on and then
laser-off. Plot of linear time data versus -ln(\selectlanguage{greek}θ\selectlanguage{english}), which was obtained
from the cooling stage (F) Photostability of OHH NPs with 5 cycles.

\textbf{3.3. Assessment of antibacterial activity}

The \emph{in vitro} antibacterial activity of OHH NPs was investigated
by bacteriostatic ring test using MRSA and \emph{K.Pneumoniae} as model
organisms. After 24 hours of culture, there was not any inhibitory area
observed in the tested bacteria after treatment with PBS or HMPB NPs. By
contrast, free ofloxacin and OHH NPs treatment groups showed obvious
inhibition zone (Fig. 3A) owing to antibacterial activity of ofloxacin.
Fig.3B and Fig.3C showed that the diameters of inhibition zones of
ofloxacin towards MRSA and \emph{K.Pneumoniae} were
\textasciitilde{}1.70 cm, \textasciitilde{}3.77 cm, respectively and the
diameters of inhibition zones of OHH NPs towards MRSA
and\emph{K.Pneumoniae} were \textasciitilde{}1.48 cm,
\textasciitilde{}3.33 cm, respectively. It should be noted that the
inhibition ring of free ofloxacin at an equivalent concentration with
loading ofloxacin of OHH NPs was slightly larger than that of OHH NPs,
which may be due to slower ofloxacin release of OHH NPs. Meanwhile, the
inhibition ring of ofloxacin for \emph{K.Pneumoniae} (Gram-negative
bacteria) was much larger than that for MRSA(Gram-positive bacteria),
which was attributed to ofloxacin with stronger antibacterial activity
towards Gram-negative bacteria. Additionally, the chemo-photothermal
synergistic bactericidal efficacy of OHH NPs was investigated using MRSA
and \emph{K.Pneumoniae}by the spread plate technique. Specifically, MRSA
and\emph{K.Pneumoniae} cells treated with different agents were diluted
10\textsuperscript{5} and 10\textsuperscript{4} times, respectively. In
Fig. 3D and 3F, the number of colonies after treatment with NIR or HMPB
NPs hardly reduced compared with control group (PBS). However, the
significant decrease of the number of bacterial colonies was observed
for HMPB NPs+NIR, ofloxacin, ofloxacin+NIR and OHH NPs treated groups.
The bacteria-killing efficacy of HMPB NPs+NIR, ofloxacin, ofloxacin+NIR
and OHH NPs treated groups reached 28.38\%, 53.90\%, 49.35\% and 27.76\%
for MRSA, respectively and it reached 51.63\%, 75.61\%, 73.52\% and
72.56\% for \emph{K.Pneumoniae} , respectively. These data demonstrated
that, in the tested dose range, both sole ofloxacin and photothermal
treatment could not eliminate all the bacterial colonies. Surprisingly,
the OHH NPs combined with NIR laser (OHH NPs+NIR) exhibited high
antibacterial efficiency towards MRSA and \emph{K.Pneumoniae} . Almost
no bacterial colonies were observed in OHH NPs+NIR treated groups and
the inactivation of bacteria was highly up to 99.99\% (Fig. 3E and 3G).
The ultrahigh antibacterial efficiency of OHH NPs+NIR could be
attributed to the effective synergistic effect of ofloxacin and PTT.
Compared with the loading rates of two different Prussian blue (Fig.S3),
we can observe that the hollow mesoporous structure can effectively
increase the loading of antibiotics. Due to the laser irradiation and
the secretion of HAase in bacterial cells, ofloxacin is released from
the nanoparticles about 80\% for 24 hours.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image4/image4}

\end{center}
\end{figure}

\textbf{Fig. 3. In vitro antibacterial activity test.} (A)Photographs of
antibacterial results of PBS(a),HMPB(b), Ofloxacin(c) and OHH NPs(d),
against MRSA and \emph{K.pneumoniae} . (B--C) Quantitative measurement
of the inhibition zones. (D,F)Photographs of the agar plates of MRSA and
Klebsiella Pneumoniae treated with PBS,HMPB,Ofloxacin and OHH NPs
with/without 808 nm light irradiation (1 W/cm\textsuperscript{2}, 5
min). (E, G) Treatment with PBS was set up as control. Effect of OHH NPs
on bacterial counts of MRSA and \emph{K.pneumoniae} . The all
concentration above of HMPB is 100\selectlanguage{greek}μ\selectlanguage{english}g/mL and ofloxacin is 10 \selectlanguage{greek}μ\selectlanguage{english}g/mL.

Next, the viability of \emph{K.pneumoniae} was further investigated by a
fluorescence staining assay based on the integrity of bacterial membrane
with HMPB,Ofloxacin and OHH at the same antibiotic concentration
(10\selectlanguage{greek}μ\selectlanguage{english}g/mL)and the same HMPB concentration (100 \selectlanguage{greek}μ\selectlanguage{english}g/mL).As shown as Fig.
4A, almost no visible red fluorescence was observed after treatment with
PBS, NIR and HMPB, indicating nearly all of the bacteria survived in
these groups. In comparison, the bacteria treated with HMPB NPs+NIR,
ofloxacin, ofloxacin+NIR and OHH NPs+NIR emitted visible red
fluorescence, which demonstrated a part of bacteria were killed.
However, the bacreria treated by OHH NPs+NIR presented the strongest red
fluorescence(Fig. 4B), suggesting most of the bacreria were
inactive\textsuperscript{38}. Additionally, SEM images of Fig. 4C
further verified that sole PBS and HMPB NPs had no antibacterial
ability, which was confirmed by the intact and smooth bacterial
membranes, while the bacterial membranes after treatment with OHH
NPs+NIR were obviously shrinking. Collectively, all of the results
provided confirmative evidence of OHH NPs having highest
bacteria-killing ability.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image5/image5}

\end{center}
\end{figure}

\textbf{Fig. 4. Observation of bacterial cell wall destruction
of\emph{K.pneumoniae}.} (A) Calcein-AM/PI staining of
\emph{K.pneumoniae}(1\selectlanguage{ngerman}×10\textsuperscript{8} CFU
mL\textsuperscript{-1})after various treatments. (normal saline, NIR,
HMPB, HMPB + NIR, Ofloxacin, Ofloxacin + NIR, OHH NPs, OHH NPs + NIR).
The net concentration of the HMPB substrate in all sample groups has
been maintained at 100 \selectlanguage{greek}μ\selectlanguage{english}g/mL. (B) The corresponding quantitative
analysis of fluorescence intensity by Image J. (C) SEM images of
\emph{K.pneumoniae} cells after various treatments.

\textbf{3.4. Antibacterial mechanism exploration}

In addition to the aforementioned results, the antibacterial mechanism
was further explored by employing DCFH-DA probe to monitor ROS
generation with different treatments on
\emph{K.pneumoniae}\textsuperscript{34, 39}. As shown in Fig. 5A and
Fig. 5B, the bacteria treated by OHH NPs +NIR exhibited the strongest
green fluorescence signal, indicating the highest level of ROS produced,
which was likely responsible for the bactericidal efficiency. As the
major energy storage molecule, ATP plays a critical role in the various
processes of physiology and pathology. Usually, ATP will decrease under
conditions of necrosis or apoptosis \textsuperscript{34, 40-42}. We thus
wish to explore if the decrease of the ATP level occurs after exposure
to OHH NPs+NIR. Fig. 5C showed HMPB NPs treatment hardly caused the
decline of ATP level in \emph{K.pneumoniae} comparing with the control
group. By comparison, a weak decrease of ATP level was caused after
incubation with ofloxacin or OHH NPs. In contrast, ATP level
dramatically declined about 92.3\% after the treatment of OHH NPs+NIR,
suggesting the best antibacterial efficacy of this strategy.
Furthermore, protein leakage from \emph{K.pneumonia} was also
investigated, which was taken as a representative indicator of cell
membrane damage \textsuperscript{43}. According to Fig. 5D, protein
leakage from \emph{K.pneumonia} of HMPB NPs-treated group was 10.07
\selectlanguage{greek}μ\selectlanguage{english}g/mL, which was similar with the control group. In comparison, protein
leakage of bacteria after exposure to ofloxacin and OHH NPs increased to
24.97 \selectlanguage{greek}μ\selectlanguage{english}g/mL and 22.06 \selectlanguage{greek}μ\selectlanguage{english}g/mL, respectively. Notably, protein leakage in
the OHH NPs+NIR-treated group was up to 42.26 \selectlanguage{greek}μ\selectlanguage{english}g/mL, which was about
5-fold that of the control group. These results indicated that the
combination of ofloxacin and PTT could severely destruct bacterial cell
membranes.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image6/image6}

\end{center}
\end{figure}

\textbf{Fig. 5. Antibacterial mechanism investigation} . (A)
Representative CLSM images to assess intracellular ROS generation
in\emph{K.pneumoniae} cells. The samples included normal saline,
HMPB(100 \selectlanguage{greek}μ\selectlanguage{english}g/mL), Ofloxacin(10 \selectlanguage{greek}μ\selectlanguage{english}g/mL), OHH NPs(100 \selectlanguage{greek}μ\selectlanguage{english}g/mL) and OHH NPs +
NIR (100 \selectlanguage{greek}μ\selectlanguage{english}g/mL) (B) The corresponding statistical histogram showing the
relative ROS level with Confocal images. (C) ATP level and (D) protein
leakage of the \emph{K.pneumoniae} suspensions treated with different
group. (E) Drug resistance development profiles of \emph{K.pneumoniae}
during serial passaging in the presence of sub-MIC dosing of PBS,
Ofloxacin, and OHH NPs. The y-axis indicates the folds of increased MIC
compared to the initial ones.

\textbf{3.5 Drug resistance evaluation}

Long-term overuse of antibiotics could easily induce drug resistance of
bacteria. Thus, we further explored whether OHH NPs with laser
assistance could reduce the occurrence of ofloxacin resistance. To
evaluate the development of bacterial resistance with different
treatments, \emph{K.pneumoniae} was continuously cultured with a
sublethal dose of ofloxacin (one-tenth of the concentration in Fig. 3A)
or OHH NPs+NIR. As shown in Fig. 5E, the MIC value of\emph{K.pneumoniae}
incubated with ofloxacin was 7 times that of initial MIC after 5
circles' incubation, suggesting serious drug resistance. On the
contrary, the MIC value of\emph{K.pneumoniae} after 5 circles' treatment
with OHH NPs+NIR was nearly same as the initial MIC. Due to the
effective synergy of OHH NPs and PTT, OHH NPs showed excellent
antibacterial ability with laser assistance. Therefore, OHH NPs combined
with laser irradiation could effectively reduce the dosage of ofloxacin
without compromising the bactericidal capability and delay the
development of drug resistance, which could be considered as a long-term
antibacterial strategy.

\textbf{3.6. In vitro dispersion of established
\emph{K.pneumoniae}biofilms}

Over 60\% of all patients' infections treated by physicians are caused
by biofilms which are of great significance for bacterial attachment and
proliferation, and extremely recalcitrant to
antimicrobials\textsuperscript{44-45}. Thus, we further studied the
potential influence for formed \emph{K.pneumoniae} biofilms mediated by
OHH NPs +NIR. As seen in Fig. 6A, comparing with the control group,
separate HMPB NPs treatment hardly showed dispersion effect on the
biofilms. In comparison, ofloxacin and OHH NPs were able to only remove
about 35\% and 26\% of the biofilm, respectively, indicating that
ofloxacin showed certain effectiveness in anti-biofilm (Fig. 6B).
However, OHH NPs with laser assistance effectively eliminated the
biofilm quantity of ~90\%, which was likely due to the excellent
synergistic effect of ofloxacin and photothermal ablation. We next
performed a fluorescence staining assay using PI to obtain 3D CLSM
images of \emph{K.pneumoniae} biofilms. It could be concluded that OHH
NPs with NIR irradiation exhibited most powerful anti-biofilms activity
from the strongest red fluorescence in Fig. 6C and Fig. 6D, which was
well consistent with the crystal violet staining assay.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image7/image7}

\end{center}
\end{figure}

\textbf{Fig. 6. In vitro anti-biofilms effect of OHH NPs} .The remaining
biofilms were quantified by crystal violet staining. Biofilms were
visualized by photographs (A) and biofilm biomass was determined by
measuring the absorbance of 590 nm. (B). (C) Confocal fluorescence
images of \emph{K.pneumoniae} biofilms. (D) The corresponding
statistical histogram showing the relative biofilm level. The group was
used as the PBS, HMPB NPs(100 \selectlanguage{greek}μ\selectlanguage{english}g/mL), ofloxacin(10 \selectlanguage{greek}μ\selectlanguage{english}g/mL), OHH NPs (100
\selectlanguage{greek}μ\selectlanguage{english}g/mL) and OHH NPs + NIR(100 \selectlanguage{greek}μ\selectlanguage{english}g/mL).

\textbf{3.7. In vitro cytotoxicity study}

The biocompatibility of agents is the most important precondition of
clinical translation. To make sure that OHH NPs could be potentially
translated for practical application, \emph{in vitro} cytotoxicity
experiments were performed with NIH-3T3 cells and HUV-EC cells.
According to Fig. 7A and Fig. S5, the viability of two cell lines
incubated with various concentrations of OHH NPs for 24 h remained at
~100\%, even at the concentration of 125 \selectlanguage{greek}μ\selectlanguage{english}g/mL, suggesting the ultra-low
cytotoxic nature of OHH NPs. The \emph{in vitro} biocompatibility of OHH
NPs was further assessed by hemolysis assay. As can be seen in Fig. 7B,
hemolytic rate after exposure to OHH NPs even at the concentration of
150 \selectlanguage{greek}μ\selectlanguage{english}g/mL was only 3.9\%, indicating good biocompatibility of OHH NPs.
In hemolysis and cytotoxicity assays (Fig. S6), it should be noted that
OHH NPs showed better biocompatibility than HMPB NPs, which was likely
attributed to the fact that HA coating with superior biocompatibility
reduced \emph{in vitro} cytotoxicity of HMPB NPs. In addition, platelet
aggregation assay displayed OHH NPs hardly caused platelet aggregation,
demonstrating their little risk of inducing thrombosis (Fig. S7).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image8/image8}

\end{center}
\end{figure}

\textbf{Fig. 7 In vitro biosafety of nanomaterials} . (A)Cell viability
and (B) hemolysis assays of HMPB, Ofloxacin, OHH NPs with different
concentrations.

\textbf{3.8. In vivo evaluation of wound healing}

We also evaluated the \emph{in vivo} healing ability of mice with
a\emph{K.pneumoniae} -infected wound utilizing the synergetic
antibacterial strategy. Female BALB/c mice with\emph{K.pneumoniae}
-infected wound on their back were used as a model for the study (Fig.
8A). Fig. 8B displayed representative photographs of wound healing after
different treatments at different time points. And traces of wound
closure for 12 days for all treatment groups were also drawn (Fig. 8C).
We observed that OHH NPs+NIR is almost healed. On day 12, the size of
infected wounds after treatment with PBS, HMPB NPs, ofloxacin,OHH NPs
and OHH NPs+NIR reduced to 35.51\%, 33.94\%, 36.82\%, 36.54\%, 17.53\%,
of the original wound area, respectively, indicating the superiority of
combination therapy for infected wound healing (Fig. 8D). Moreover, the
insignificant body weight change of mice demonstrated all treatment
groups with the low toxicity (Fig. 8E). In addition, the\emph{in vivo}
antibacterial effect of all treatment groups was also assessed. As shown
in Fig. 8F and 8G, the combination of OHH NPs and PTT exhibited best
antibacterial effect for \emph{K.pneumoniae} -infected wound comparing
with all single-mode therapies, which was in accord with the \emph{in
vitro} antibacterial test in Fig. 3. Besides, the temperature of the
wound region treated with OHH NPs increased to \textasciitilde{}52 \selectlanguage{ngerman}°C
after irradiation for 5 min, the temperature of group PBS did not change
significantly. (Fig. S8).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image9/image9}

\end{center}
\end{figure}

\textbf{Fig. 8. Macroscopic evaluation and histological analysis of
wound models in vivo.} (A) Scheme for in vivo test including the
establishment of \emph{K.pneumoniae} infection model and subsequent
treatment regime. (B) Changes in mice body weight during the treatment.
(C) Representative photographs of wounds at day 0, 4, 8, and 12 and (D)
wound closure rates. (E) Representative photographs of bacteria colonies
derived from the infected sites of mice with different treatment as
indicated. (F) Corresponding quantitative data of bacterial colonies in
(E). (G) Wound area traces of each group during 12 days.

The wound closure efficacy after different treatments was also assessed
by performing histological analysis of wound tissues. As shown in Fig.
9A, mass inflammatory cells were observed in PBS, HMPB, Ofloxacin, and
OHH NPs groups, suggesting the wound recovery was incomplete. However,
the wounds of OHH NPs+NIR showed almost as complete as normal tissue
without obvious inflammation. Furthermore, the collagen deposition of
wound area was investigated by Masson's trichrome staining. Fig. 9B
showed the regenerated collagen fiber stained with blue color in OHH
NPs+NIR-treated group was continuous and more obvious than that in other
treated groups, which reflected better recovery of combination therapy.
All of the results confirmed the best healing efficiency of OHH NPs+NIR
strategy.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/imagf0/imagf0}

\end{center}
\end{figure}

\textbf{Fig. 9.} \textbf{Histologic analysis of infected tissues after
12 days of treatments .} (A) HE staining;(B) Masson staining.

\textbf{3.9. In vivo biological safety study}

In order to evaluate the in vivo potential toxicity of OHH NPs+NIR,
biochemical analysis of all mice with different treatments was performed
when wound healing experiment was completed. Fig. 10A-D showed that
there were no obvious differences among all groups in total white blood
cell (WBC), total red blood cells (RBC), hemoglobin (HGB) and blood
platelet (PLT) counts. Meanwhile, no abnormalities were found in key
functional hepatic and kidney indicators, such as ALT, AST, UREA and
CREA (Fig. 10E-H). In addition, H\&E staining images of the major organs
including heart, liver, spleen, kidney, and lung confirmed normal tissue
structures without any visible organ damage. All these results strongly
demonstrated our developed therapeutic strategy of OHH NPs+NIR with the
superior biosafety for in vivo infected wound healing therapy.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/imagf1/imagf1}

\end{center}
\end{figure}

\textbf{Fig. 10. Toxicity evaluation of nanoparticles in vivo.} (A-D)
Blood biochemistry data WBC, RBC, HGB and PLT (E-H) Blood biochemistry
data including liver and kidney function markers: ALT, AST, UREA and
CREA. (I) Representative H\&E stained slices of major organs.

\textbf{4. Conclusion}

In summary, the OHH NPs nanoplatform was fabricated for
antibiotic-photothermal ablation of \emph{K.pneumoniae} bacteria. The
integrated nanoplatform could efficiently adhere onto\emph{K.pneumoniae}
bacteria and damage bacterial membrane by subsequent PTT under NIR
irradiation. The exploration of the antibacterial mechanism shows that
the powerful bactericidal ability of the cooperative system and the
inhibition of drug resistance can be attributed to the generation of
ROS, which destroys the integrity of cell membranes, reduces ATP and
disrupts bacterial metabolism. The outstanding bactericidal capability
of the OHH NPs nanoplatform under NIR irradiation was also confirmed by
an in vivo\emph{K.pneumoniae} -infected model on mice. OHH NPs
nanoplatform combined with antibiotic-photothermal therapy can be
considered as a very promising strategy to effectively eliminate
bacteria and inhibit the development of drug resistance.

\textbf{Acknowledgement}

This work was partially supported by the Natural Science Foundation of
Hunan Province (2020JJ4005, 2021JJ3016), the Opening Foundation of Key
Laboratory of Study and Discovery of Small Targeted Molecules of Hunan
Province(2019CG03), the Opening Foundation of School of Medicine, Hunan
Normal University(KF2021023).

\textbf{Conflicts of interest}

The authors declare that they have no conflict of interest.

\textbf{References}

(1) Taubes, G., The Bacteria Fight Back. \emph{Science}
\textbf{2008,}\emph{321} (5887), 356-361.

(2) Levy, S. B.; Marshall, B., Antibacterial resistance worldwide:
causes, challenges and responses. \emph{Nat Med} \textbf{2004,}\emph{10}
(12 Suppl), S122-9.

(3) Hartmann, M.; Betz, P.; Sun, Y.; Gorb, S. N.; Lindhorst, T. K.;
Krueger, A., Saccharide-modified nanodiamond conjugates for the
efficient detection and removal of pathogenic bacteria.
\emph{Chemistry}\textbf{2012,} \emph{18} (21), 6485-92.

(4) Kieron; M.; G.; O'Connell; James; T.; Hodgkinson; Hannah; F.; Sore,
Combating Multidrug-Resistant Bacteria: Current Strategies for the
Discovery of Novel Antibacterials. \emph{Angewandte Chemie International
Edition} \textbf{2013,} \emph{52} (41), 10706-10733.

(5) Morgan, D.; Okeke, I. N.; Laxminarayan, R.; Perencevich, E. N.;
Weisenberg, S., Non-prescription antimicrobial use worldwide: a
systematic review. \emph{Lancet Infectious Diseases}
\textbf{2011,}\emph{11} (9), 692-701.

(6) Michaelidou, M.; Karageorgos, S. A.; Tsioutis, C., Antibiotic Use
and Antibiotic Resistance: Public Awareness Survey in the Republic of
Cyprus. \emph{Antibiotics (Basel)} \textbf{2020,} \emph{9} (11).

(7) Qing, G.; Zhao, X.; Gong, N.; Chen, J.; Liang, X. J.,
Thermo-responsive triple-function nanotransporter for efficient
chemo-photothermal therapy of multidrug-resistant bacterial
infection.\emph{Nature Communications} \textbf{2019,} \emph{10} (1),
1-12.

(8) Sun, P.; Zhang, Y.; Ran, X.; Liu, C.; Wang, Z.; Ren, J.; Qu, X.,
Phytochemical-encapsulated nanoplatform for ``on-demand'' synergistic
treatment of multidrug-resistant bacteria. \emph{Nano
Research}\textbf{2018,} \emph{11} (7), 3762-3770.

(9) Zhang, Y.; Sun, P.; Zhang, L.; Wang, Z.; Wang, F.; Dong, K.; Liu,
Z.; Ren, J.; Qu, X., Silver-Infused Porphyrinic Metal-Organic Framework:
Surface-Adaptive, On-Demand Nanoplatform for Synergistic Bacteria
Killing and Wound Disinfection. \emph{Advanced Functional
Materials}\textbf{2019,} \emph{29} (11).

(10) Wang, Y.; Ding, X.; Chen, Y.; Guo, M.; Zhang, Y.; Guo, X.; Gu, H.,
Antibiotic-loaded, silver core-embedded mesoporous silica nanovehicles
as a synergistic antibacterial agent for the treatment of drug-resistant
infections. \emph{Biomaterials} \textbf{2016,} \emph{101} , 207-216.

(11) Gao, G.; Jiang, Y. W.; Jia, H. R.; Wu, F. G., Near-infrared
light-controllable on-demand antibiotics release using thermo-sensitive
hydrogel-based drug reservoir for combating bacterial
infection.\emph{Biomaterials} \textbf{2019,} \emph{188} , 83-95.

(12) Wu, Y.; Song, Z.; Wang, H.; Han, H., Endogenous stimulus-powered
antibiotic release from nanoreactors for a combination therapy of
bacterial infections. \emph{Nat Commun} \textbf{2019,} \emph{10} (1),
4464.

(13) Tao, B.; Lin, C.; Yuan, Z.; He, Y.; Cai, K., Near infrared
light-triggered on-demand Cur release from Gel-PDA@Cur composite
hydrogel for antibacterial wound healing. \emph{Chemical Engineering
Journal} \textbf{2020,} \emph{403} , 126182.

(14) Zhao, Y.; Dai, X.; Wei, X.; Yu, Y.; Chen, X.; Zhang, X.; Li, C.,
Near-Infrared Light-Activated Thermosensitive Liposomes as Efficient
Agents for Photothermal and Antibiotic Synergistic Therapy of Bacterial
Biofilm. \emph{ACS Appl Mater Interfaces} \textbf{2018,} \emph{10} (17),
14426-14437.

(15) J. Ouyang, R. Y. L., W. Chen, Z. Liu, Q. Xu, K. Zeng, L. Deng, L.
Shen, Y.N. Liu,, A black phosphorus based synergistic antibacterial
platform against drug resistant bacteria. \emph{J. Mater. Chem.
B}\textbf{2018,} \emph{6} , 6302-6310.

(16) Wang X, S. K., Tan L, Liu X, Cui Z, Jing D, Yang X, Liang Y, Li Z,
Zhu S, Yeung KWK, Zheng D, Wu S, Rapid and highly effective noninvasive
disinfection by hybrid Ag/CS@MnO\textsubscript{2} nanosheets using
near-infrared light{[}J{]}. \emph{ACS Appl. Mater.
Interfaces}\textbf{2019,} \emph{11} (16), 15014-15027.

(17) Yang, Y.; He, P.; Wang, Y.; Bai, H.; Wang, S.; Xu, J.-F.; Zhang,
X., Supramolecular Radical Anions Triggered by Bacteria In Situ for
Selective Photothermal Therapy. \emph{Angewandte Chemie International
Edition} \textbf{2017,} \emph{56} (51), 16239-16242.

(18) Gao, Q.; Zhang, X.; Yin, W.; Ma, D.; Xie, C.; Zheng, L.; Dong, X.;
Mei, L.; Yu, J.; Wang, C.; Gu, Z.; Zhao, Y., Functionalized MoS2
Nanovehicle with Near-Infrared Laser-Mediated Nitric Oxide Release and
Photothermal Activities for Advanced Bacteria-Infected Wound
Therapy.\emph{Small} \textbf{2018,} \emph{14} (45), 1802290.

(19) Yang, Y.; Ma, L.; Cheng, C.; Deng, Y.; Huang, J.; Fan, X.; Nie, C.;
Zhao, W.; Zhao, C., Nonchemotherapic and Robust Dual-Responsive
Nanoagents with On-Demand Bacterial Trapping, Ablation, and Release for
Efficient Wound Disinfection. \emph{Advanced Functional
Materials}\textbf{2018,} \emph{28} (21), 1705708.

(20) Hu, D.; Li, H.; Wang, B.; Ye, Z.; Lei, W.; Jia, F.; Jin, Q.; Ren,
K.-F.; Ji, J., Surface-Adaptive Gold Nanoparticles with Effective
Adherence and Enhanced Photothermal Ablation of Methicillin-Resistant
Staphylococcus aureus Biofilm. \emph{ACS Nano} \textbf{2017,}
\emph{11}(9), 9330-9339.

(21) Ray, P. C.; Khan, S. A.; Singh, A. K.; Senapati, D.; Fan, Z.,
Nanomaterials for targeted detection and photothermal killing of
bacteria. \emph{Chemical Society Reviews} \textbf{2012,} \emph{41} (8),
3193.

(22) Gao, Q.; Zhang, X.; Yin, W.; Ma, D.; Xie, C.; Zheng, L.; Dong, X.;
Mei, L.; Yu, J.; Wang, C.; Gu, Z.; Zhao, Y., Functionalized MoS2
Nanovehicle with Near-Infrared Laser-Mediated Nitric Oxide Release and
Photothermal Activities for Advanced Bacteria-Infected Wound
Therapy.\emph{Small} \textbf{2018,} \emph{14} (45), e1802290.

(23) Yuwen, L.; Sun, Y.; Tan, G.; Xiu, W.; Zhang, Y.; Weng, L.; Teng,
Z.; Wang, L., MoS2@polydopamine-Ag nanosheets with enhanced
antibacterial activity for effective treatment of Staphylococcus aureus
biofilms and wound infection. \emph{Nanoscale} \textbf{2018,}
\emph{10}(35), 16711-16720.

(24) J. Sun, L. S., Y. Fan, L. Tian, S. Luan, S. Niu, L. Ren, W. Ming,
J. Zhao, Synergistic photodynamic and photothermal antibacterial
nanocomposite membrane triggered by single NIR light source. \emph{ACS
Appl Mater Interfaces} \textbf{2019,} \emph{11} (30), 26581-26589.

(25) Rubio-Ruiz, B.; Perez-Lopez, A. M.; Bray, T. L.; Lee, M.; Serrels,
A.; Prieto, M.; Arruebo, M.; Carragher, N. O.; Sebastian, V.;
Unciti-Broceta, A., High-Precision Photothermal Ablation Using
Biocompatible Palladium Nanoparticles and Laser Scanning
Microscopy.\emph{ACS Appl Mater Interfaces} \textbf{2018,} \emph{10}
(4), 3341-3348.

(26) Wang, H.; Zhao, B.; Dong, W.; Zhong, Y.; Zhang, X.; Gong, Y.; Zhan,
R.; Xing, M.; Zhang, J.; Luo, G.; Qian, W., A dual-targeted platform
based on graphene for synergistic chemo-photothermal therapy against
multidrug-resistant Gram-negative bacteria and their
biofilms.\emph{Chemical Engineering Journal} \textbf{2020,} \emph{393} ,
124595.

(27) Zeng, X.; Luo, M.; Liu, G.; Wang, X.; Tao, W.; Lin, Y.; Ji, X.;
Nie, L.; Mei, L., Polydopamine-Modified Black Phosphorous Nanocapsule
with Enhanced Stability and Photothermal Performance for Tumor
Multimodal Treatments. \emph{Advanced science} \textbf{2018,}
\emph{5}(10), 1800510.

(28) Mei, Z.; Gao, D.; Hu, D.; Zhou, H.; Ma, T.; Huang, L.; Liu, X.;
Zheng, R.; Zheng, H.; Zhao, P.; Zhou, J.; Sheng, Z., Activatable NIR-II
photoacoustic imaging and photochemical synergistic therapy of MRSA
infections using miniature Au/Ag nanorods.
\emph{Biomaterials}\textbf{2020,} \emph{251} , 120092.

(29) C. Tong, X. Z., Y. Yang, X. Liu, G. Zhong, C. Xiao, B. Liu, W.
Wang, X. Yang, PB@PDA@Ag nanosystem for synergistically eradicating MRSA
and accelerating diabetic wound healing assisted with laser
irradiation.\emph{Biomaterials} \textbf{2020,} \emph{243} , 119936.

(30) Liu B, W. W., Fan J, Long Y, Xiao F, et al, RBC membrane
camouflaged prussian blue nanoparticles for gamabutolin loading and
combined chemo/photothermal therapy of breast cancer. \emph{Biomaterials
217} , 119301.

(31) Cai, X.; Jia, X.; Gao, W.; Zhang, K.; Ma, M.; Wang, S.; Zheng, Y.;
Shi, J.; Chen, H., A Versatile Nanotheranostic Agent for Efficient
Dual-Mode Imaging Guided Synergistic Chemo-Thermal Tumor
Therapy.\emph{Advanced Functional Materials} \textbf{2015,} \emph{25}
(17), 2520-2529.

(32) Zhou, J.; Li, M.; Hou, Y.; Luo, Z.; Chen, Q.; Cao, H.; Huo, R.;
Xue, C.; Sutrisno, L.; Hao, L.; Cao, Y.; Ran, H.; Lu, L.; Li, K.; Cai,
K., Engineering of a Nanosized Biocatalyst for Combined Tumor Starvation
and Low-Temperature Photothermal Therapy. \emph{ACS Nano}
\textbf{2018,}\emph{12} (3), 2858-2872.

(33) Fan, J.; Liu, B.; Long, Y.; Wang, Z.; Tong, C.; Wang, W.; You, P.;
Liu, X., Sequentially-targeted biomimetic nano drug system for
triple-negative breast cancer ablation and lung metastasis
inhibition.\emph{Acta Biomater} \textbf{2020,} \emph{113} , 554-569.

(34) Tong, C.; Zhong, X.; Yang, Y.; Liu, X.; Zhong, G.; Xiao, C.; Liu,
B.; Wang, W.; Yang, X., PB@PDA@Ag nanosystem for synergistically
eradicating MRSA and accelerating diabetic wound healing assisted with
laser irradiation. \emph{Biomaterials} \textbf{2020,} \emph{243} ,
119936.

(35) Liu, B.; Wang, W.; Fan, J.; Long, Y.; Xiao, F.; Daniyal, M.; Tong,
C.; Xie, Q.; Jian, Y.; Li, B.; Ma, X.; Wang, W., RBC membrane
camouflaged prussian blue nanoparticles for gamabutolin loading and
combined chemo/photothermal therapy of breast cancer.\emph{Biomaterials}
\textbf{2019,} \emph{217} , 119301.

(36) Xiao, F.; Fan, J.; Tong, C.; Xiao, C.; Wang, Z.; Liu, B.; Daniyal,
M.; Wang, W., An erythrocyte membrane coated mimetic nano-platform for
chemo-phototherapy and multimodal imaging. \emph{RSC
Advances}\textbf{2019,} \emph{9} (48), 27911-27926.

(37) Zhong, X.; Tong, C.; Liu, T.; Li, L.; Liu, X.; Yang, Y.; Liu, R.;
Liu, B., Silver nanoparticles coated by green graphene quantum dots for
accelerating the healing of MRSA-infected wounds. \emph{Biomaterials
Science} \textbf{2020,} \emph{8} (23), 6670-6682.

(38) Wang, L.; Yang, J.; Yang, X.; Hou, Q.; Liu, S.; Zheng, W.; Long,
Y.; Jiang, X., Mercaptophenylboronic Acid-Activated Gold Nanoparticles
as Nanoantibiotics against Multidrug-Resistant Bacteria. \emph{ACS Appl
Mater Interfaces} \textbf{2020,} \emph{12} (46), 51148-51159.

(39) Li, Y.; Gao, Z.; Zhang, Y.; Chen, F.; An, P.; Wu, H.; You, C.; Sun,
B., MOF-shielded and glucose-responsive ultrasmall silver nano-factory
for highly-efficient anticancer and antibacterial therapy.\emph{Chemical
Engineering Journal} \textbf{2021,} \emph{416} , 127610.

(40) Tong, C.; Li, L.; Xiao, F.; Fan, J.; Zhong, X.; Liu, X.; Liu, B.;
Wu, Z.; Zhou, J., Daptomycin and AgNP co-loaded rGO nanocomposites for
specific treatment of Gram-positive bacterial infection in vitro and in
vivo. \emph{Biomaterials Science} \textbf{2019,} \emph{7} (12),
5097-5111.

(41) Tong, C.; Zou, W.; Ning, W.; Fan, J.; Li, L.; Liu, B.; Liu, X.,
Synthesis of DNA-guided silver nanoparticles on a graphene oxide
surface: enhancing the antibacterial effect and the wound healing
activity. \emph{RSC Advances} \textbf{2018,} \emph{8} (49), 28238-28248.

(42) Cui, Y.; Zhao, Y.; Tian, Y.; Zhang, W.; Lu, X.; Jiang, X., The
molecular mechanism of action of bactericidal gold nanoparticles on
Escherichia coli. \emph{Biomaterials} \textbf{2012,} \emph{33} (7),
2327-33.

(43) Zhao, R.; Kong, W.; Sun, M.; Yang, Y.; Liu, W.; Lv, M.; Song, S.;
Wang, L.; Song, H.; Hao, R., Highly Stable Graphene-Based Nanocomposite
(GO-PEI-Ag) with Broad-Spectrum, Long-Term Antimicrobial Activity and
Antibiofilm Effects. \emph{ACS Appl Mater Interfaces}
\textbf{2018,}\emph{10} (21), 17617-17629.

(44) Hu, D.; Zou, L.; Yu, W.; Jia, F.; Han, H.; Yao, K.; Jin, Q.; Ji,
J., Relief of Biofilm Hypoxia Using an Oxygen Nanocarrier: A New
Paradigm for Enhanced Antibiotic Therapy. \emph{Adv Sci
(Weinh)}\textbf{2020,} \emph{7} (12), 2000398.

(45) Ivanova, A.; Ivanova, K.; Tied, A.; Heinze, T.; Tzanov, T.,
Layer-By-Layer Coating of Aminocellulose and Quorum Quenching Acylase on
Silver Nanoparticles Synergistically Eradicate Bacteria and Their
Biofilms. \emph{Advanced Functional Materials} \textbf{2020,}
\emph{30}(24).

\selectlanguage{english}
\FloatBarrier
\end{document}

