Prospective Observational Study of Perioperative Biomarkers and Outcomes in Deep Inferior Epigastric Perforator (DIEP) Flap Breast Reconstruction
Conditions: Breast Neoplasms, Ischemia-Reperfusion Injury, Postoperative Complications, Wound Healing, Oxidative Stress, Inflammation
Sponsor: Hubei Cancer Hospital

COVER PAGE 
Official Title: Mn/QD-SAC–Guided NIR-IIb Imaging and Antioxidant Therapy for 
Ischemia–Reperfusion Injury in DIEP Flaps  
Document Type: Study Protocol and Statistical Analysis Plan  
ClinicalTrials.gov Identifier (NCT Number): NCTXXXXXXXX  
Sponsor/Institution: Hubei Cancer Hospital 
Study Location: Breast Surgery Department, Hubei Cancer Hospital, Wuhan, China 
Principal Investigator: Xinhong Wu, MD  
Department: Breast Surgery 
Document Date: 11 Sep 2025  
Version: v1.0  
 
 
 
 
 
 
 
 
 
 
 

1. SYNOPSIS 
（1）Background: Deep inferior epigastric perforator (DIEP) flap breast reconstruction 
is valuable but prone to microcirculatory instability and ischemia–reperfusion injury 
(I/R). NIR-IIb (1500–1700 nm) imaging offers deeper penetration and higher SNR than 
conventional ICG-NIR, potentially enabling earlier detection of perfusion deficits. A 
manganese-modified single-atom catalyst on NIR-IIb quantum dots (Mn/QD-SAC) 
combines deep-tissue imaging with broad-spectrum ROS-scavenging enzyme-mimetic 
activity to mitigate I/R damage. 
（2）Hypothesis: Intraoperative and postoperative application of Mn/QD-SAC will 
enable earlier and deeper detection of perfusion deficits via NIR-IIb imaging and 
attenuate ROS-driven inflammatory cascades, reducing adipose necrosis and partial 
flap loss. 
（3）Design: Preclinical materials development; in vitro mechanistic assays; small-
animal DIEP-like flap I/R model for imaging/therapy; and an observational, minimal-
risk human biospecimen sub-study (peripheral blood from DIEP patients) to assess 
circulating biomarkers. 
（4）Primary Endpoints: Flap survival rate at postoperative Day 7; NIR-IIb SNR and 
correlation with final flap survival area. 
（5）Secondary Endpoints: Materials properties; in vitro cell survival/apoptosis/ROS 
and cytokines; in vivo oxidative stress, inflammation, apoptosis, histology; safety and 
biodistribution. 
（6）Statistics: ANOVA/appropriate nonparametrics for group comparisons; repeated-
measures ANOVA; Kaplan–Meier for flap survival; Pearson/Spearman for correlations. 
Two-sided alpha 0.05. 
2. BACKGROUND AND RATIONALE  

DIEP flap reconstruction avoids implants and offers natural contour, but intraoperative 
microcirculatory instability and I/R significantly affect outcomes, with reported double-
digit rates of fat necrosis or partial flap loss. I/R triggers a burst of ROS (mitochondrial 
and enzyme sources such as xanthine oxidase and NOX), causing lipid peroxidation, 
apoptosis, endothelial dysfunction, and inflammation. Conventional ICG-NIR imaging 
assists real-time perfusion assessment, yet is constrained by limited depth and 
autofluorescence. NIR-IIb improves penetration and SNR, better capturing 
subcutaneous fat and deep microvasculature. Mn/QD-SAC integrates imaging with 
therapy: single-atom Mn active sites provide ROS-scavenging enzyme-mimetic activity 
while QDs enable NIR-IIb emission. Pilot evidence in a murine brain injury model 
supports feasibility for ROS suppression, inflammation inhibition, and tissue repair, 
warranting systematic evaluation in flap I/R models. 
3. OBJECTIVES Primary 
（1）Develop and validate in vivo NIR-IIb imaging performance (sensitivity, timing, 
quantification) of Mn/QD-SAC in DIEP flap I/R and compare to ICG-NIR, laser 
Doppler/speckle, and tissue oxygenation measurements. 
（2 ）Demonstrate antioxidant, anti-inflammatory, and anti-apoptotic effects and 
mechanisms (e.g., Nrf2/HO-1, NF-κB, JAK/STAT). 
（3）Establish a feasible workflow in murine flap I/R and a clinical preclinical human 
biospecimen sub-study (DIEP patient peripheral blood) to inform future trials and 
safety assessment. 
4. STUDY DESIGN OVERVIEW 
（1 ）Part A: Materials synthesis and characterization of Mn/QD-SAC 
(physicochemical, optical, catalytic). 
（2）Part B: In vitro mechanistic assays in endothelial, adipose/mesenchymal, and 

immune cells under hypoxia–reoxygenation (H/R). 
（3）Part C: In vivo murine DIEP-like flap I/R model to test NIR-IIb imaging 
performance and therapeutic efficacy; safety and biodistribution. 
（4）Part D: Human biospecimen sub-study (observational) in DIEP patients for 
peripheral blood biomarkers at predefined perioperative timepoints; no investigational 
product administered to humans in this protocol. 
5. METHODS  
5.1Materials Synthesis and Characterization  
（1 ）Objective: Produce reproducible, stable Mn/QD-SAC and complete 
physicochemical, optical, and functional characterization. 
（2）QD preparation: Ag2Te/Ag2Se-based QDs with controlled size for NIR-IIb 
emission (1500–1700 nm). 
（3）Mn single-atom incorporation: Wet-chemical loading/thermal treatment to achieve 
isolated Mn sites on QD surfaces; confirm by HAADF-STEM. 
（4）Surface modification: mPEG-SH or DSPE-PEG ligand exchange/encapsulation 
to reduce protein adsorption and enhance circulation; measure zeta potential and 
solubility.  
（5 ）Characterization: TEM/HAADF-STEM, XPS, XRD, UV–Vis–NIR, and 
fluorescence spectroscopy (QY, emission peak), ESR for radical scavenging, and 
stability in serum/salt/protease.  
（6）In vitro catalytic activity: SOD-like and CAT-like assays; ESR trapping for •OH 
and O2•–. 
5.2 In Vitro Models and Readouts  

（1）Cell types: 
Endothelial: HUVEC or HDMEC (barrier function, NO production, apoptosis). 
Adipose/mesenchymal: hADSC; adipogenic 3T3-L1 (fat tissue susceptibility to I/R). 
Immune: RAW264.7 (M1/M2 polarization, cytokine secretion).  
（2）H/R model: 1% O2 hypoxia for 4–8 h followed by reoxygenation (21% O2); 
monitor ROS dynamics and viability.  
（3）Groups: Vehicle; QD without Mn; Mn/QD-SAC low/medium/high doses; positive 
control NAC (e.g., 5 mM).  
（4）Readouts: Cell viability (CCK-8 or CellTiter-Glo), apoptosis (Annexin V/PI), 
ROS (DCFH-DA, MitoSOX), mitochondrial membrane potential (JC-1), cytokines 
(ELISA: TNF-α, IL-6, IL-1β), and Western blot (Nrf2, HO-1, p-NF-κB, p-STAT3, 
iNOS/Arg1). 
5.3 In Vivo DIEP-like Flap I/R Model  
（1）Model: Murine abdominal perforator-based pedicled or free flap; define ischemia 
onset; two ischemia durations (2 h and 4 h) for mild/moderate injury.  
（2）Randomization (n≈8/group): Sham; I/R+PBS; I/R+QD (no Mn); I/R+Mn/QD-
SAC low; I/R+Mn/QD-SAC medium; I/R+NAC.  
（3）Dosing (IV tail vein): Imaging 1 mg/kg; therapeutic 5 mg/kg; high-dose 10 mg/kg 
for toxicity arm.  
（4）Timing: Administer at reperfusion; optional repeat at 6 h and 24 h.  
（5）Imaging: NIR-IIb intraoperative and at 0 min, 1 h, 6 h, 24 h, 72 h, Day 7, Day 14; 
record ROI intensity, perfused area, time-to-peak, SNR. Cross-validate with ICG-NIR 

(intraop) and laser Doppler/speckle.  
（6）Endpoints: 
Functional/imaging: Flap survival area (Day 7–14), predictive accuracy of imaging vs 
final survival. 
Molecular/tissue: ROS and oxidative stress (MDA, 8-OHdG, 4-HNE), GSH/GSSG; 
cytokines (tissue/serum ELISA); IHC (CD31, MPO, MMP-9); TUNEL; Western blot 
(Nrf2, HO-1, p–NF-κB, p-STAT3). 
Safety/biodistribution: CBC; serum chemistries (ALT/AST, creatinine); histopathology 
(liver/kidney/spleen/lung/heart); ICP-MS of Ag and Mn in blood/organs.  
（7）Blinding: Randomization of animals; imaging and histology analyses performed 
blinded to group. 
5.4 Human Biospecimen Sub-study (Observational)  
（1）Population: Female, 18–70, undergoing immediate DIEP flap reconstruction after 
breast cancer surgery; n≈30 convenience sample.  
（2）Consent/IRB: Written informed consent before any procedures. Protocol and ICF 
approved by the hospital ethics committee; amendments submitted for approval.  
（3）Data and samples: 
De-identified clinical data: age, BMI, operative time, ischemia duration, clinical flap 
viability, complications. 
Peripheral blood draws (10 mL each; serum tube + EDTA) at: pre-op baseline; 
postoperative 0 h (immediate), 6 h, 24 h, and 72 h.  
Analytes: 
Hematology/coagulation: CBC, PT, APTT, fibrinogen, D-dimer. 

Inflammation/oxidative stress: IL-6, IL-1β, TNF-α, IL-8/IL-10/IL-18, MDA, 
8-iso-PGF2α, SOD, CAT, GSH-Px, total antioxidant capacity. 
Injury/repair: Lactate, LDH, HMGB1, CK-MB, vWF, VEGF, HIF-1α.  
Notes: Results are for research only and not used for clinical decision-making unless 
an incidental clinically actionable finding is identified and IRB-approved for disclosure. 
6. OUTCOME MEASURES  
Primary 
（1）In vivo therapeutic efficacy: Flap survival rate (%) at Day 7. 
（2）In vivo imaging: NIR-IIb SNR and correlation with final flap survival area. 
Secondary 
（1）Materials: Quantum yield; ROS scavenging efficiency; SOD-like and CAT-like 
activities. 
（2）In vitro: Cell viability; apoptosis; ROS levels; cytokines; pathway proteins. 
（3）In vivo biology: MDA, SOD, GSH-Px; cytokines (TNF-α, IL-6, IL-1β, IL-10); 
histology scores; TUNEL; WB/IHC markers. 
（4 ）Safety: Body weight; CBC; chemistries; organ histopathology; elemental 
distribution/clearance (ICP-MS). 
7. STATISTICAL ANALYSIS PLAN  
（1）Analysis populations: All randomized animals analyzed per assigned group. All 
processed biospecimens are included if predefined QC passes.  
（2）Descriptive statistics: Mean±SD or median[IQR] as appropriate; 95% CIs where 
informative.  

（3）Group comparisons: One-way ANOVA with post hoc tests (e.g., LSD-t) for 
parametric data; Kruskal–Wallis with Dunn’s post hoc for nonparametric data.  
（4）Repeated measures: Repeated-measures ANOVA with appropriate covariance 
structure; Greenhouse–Geisser correction if sphericity is violated.  
（5）Survival analyses: Kaplan–Meier for flap survival; log-rank test for group 
differences.  
（6）Correlations: Pearson (parametric) or Spearman (nonparametric).  
（7）Multiplicity: Control via planned comparisons; exploratory nature acknowledged.  
（8）Sample size: Preclinical exploratory study; no formal power calculation. Group 
size n≈8 informed by prior effect sizes and feasibility. Human sub-study (n≈30) is 
hypothesis-generating.  
（9）Software: SPSS v26.0 and GraphPad Prism v9.0.  
（10）Significance: Two-sided alpha 0.05. 
8. DATA MANAGEMENT AND QUALITY ASSURANCE 
（1）Data capture: De-identified unique study IDs. Source data is maintained securely. 
Double data entry or programmatic QC where feasible. 
（2）Monitoring: Periodic internal review of consent documentation, sample logs, 
imaging raw files, and analysis scripts. 
（3）Archiving: Retain per regulations; controlled destruction or extended retention 
with IRB approval. 
9. PROTECTION OF HUMAN SUBJECTS AND PRIVACY 

（1）Consent and ethics: Clear explanation of purpose, procedures, risks/benefits; 
voluntary participation; IRB approval before initiation; amendments submitted for 
review. 
（2）Privacy: Coded IDs at collection; linkage file stored separately with restricted 
access. Publications use aggregate or anonymized data only. 
（3）Data security: Encrypted institutional servers; role-based access; regular backups; 
no public cloud for raw PHI. Paper records are stored locked. 
（4）Biospecimens: Labeled by ID only; used solely for prespecified assays. Residual 
samples require new IRB approval and/or renewed consent, or are destroyed per 
biosafety procedures. 
（5）Feedback: Individual lab results are not returned for clinical use unless incidental, 
clinically actionable findings are confirmed and IRB-approved for disclosure. 
10. RISK–BENEFIT ASSESSMENT 
（1）Animals: Risks from surgery/anesthesia/agents mitigated by veterinary oversight 
and humane endpoints; analgesia per IACUC. 
（2）Human sub-study: Minimal risk from phlebotomy; no investigational product 
administered to humans in this protocol. Potential societal benefit from improved 
understanding of I/R biomarkers and imaging strategies. 
11.ETHICAL AND REGULATORY COMPLIANCE 
（1）Conduct per Declaration of Helsinki, International Ethical Guidelines, and 
applicable national regulations and institutional policies. 
（2）Animal studies per institutional IACUC standards and relevant guidelines. 
12.DISSEMINATION AND DATA SHARING 

（1）Results will be disseminated in peer-reviewed publications and conferences. 
（2）IPD sharing plan: No sharing of IPD is planned due to patient privacy and ethical 
considerations, lack of consent for third-party data sharing, institutional/regulatory 
restrictions, and limited resources for secure de-identification/hosting. Aggregate 
results will be available in publications or upon request. 
13.Administrative Information 
Version: v1.0 
Version Date: 11 Sep 2025. 
Department: Breast Surgery, Hubei Cancer Hospital 
Sponsor/Institution: Hubei Cancer Hospital