Impact of Conjugation Chemistry on the Pharmacokinetics of Peptide–Polymer Conjugates in a Model of Traumatic Brain Injury

Abstract

Traumatic brain injury (TBI) remains a leading cause of long-term disability and mortality; however, there are no effective therapies to mitigate secondary injury and long-term neurological impairments. After the initial mechanical insult, there is a secondary injury that leads to neuroinflammation and blood–brain barrier (BBB) disruption, both of which are linked to changes in the extracellular matrix (ECM). A short peptide sequence, CAQK (Cys-Ala-Gln-Lys), targets upregulated ECM proteoglycans after TBI and has exhibited therapeutic properties in preclinical TBI studies. However, like many peptides, CAQK has poor pharmacokinetics, with rapid systemic clearance limiting its therapeutic potential. To overcome these limitations, we investigated a peptide–polymer conjugate using a poly­(ethylene glycol) (PEG) scaffold to improve the peptide pharmacokinetics of CAQK. We synthesized materials using two conjugation chemistries, maleimide–thiol Michael-type addition and dibenzocyclooctyne (DBCO)-azide strain-promoted azide–alkyne cycloaddition. The impact of linker selection on biodistribution and clearance was distinct. We first showed that conjugation of CAQK to PEG, irrespective of linkers, significantly extended the peptide's blood half-life by 90-fold and increased brain accumulation. In the analysis of off-target organs, we observed longer retention of DBCO conjugates in the liver, kidney, and spleen compared to maleimide conjugates. Given the high incidence of TBI in populations such as military personnel and athletes, we explored whether our long-circulating material could be given as a prophylaxis. We demonstrated the accumulation of 4.5%ID/g CAQK in the injured brain when the conjugate was delivered prophylactically 24 h before injury. Our work underscores the advantage of long-circulating peptide–polymer conjugates in the context of TBI and the impact of conjugation chemistry on pharmacokinetics.

Introduction

Traumatic brain injury (TBI) is a significant public health concern, affecting both civilian and military populations. − According to the Defense Medical Surveillance System (DMSS), from 2000 through the third quarter of 2022, over 510,000 U.S. service members were diagnosed with TBI, with mild cases comprising approximately 82% of these diagnoses. TBI is a complex neurological condition characterized by both primary and secondary injury mechanisms. The primary injury results from the initial mechanical insult, causing direct neuronal and glial damage and transient dysregulation of the blood–brain barrier (BBB). This is followed by the secondary injury phase, a cascade of biochemical and molecular events, including inflammation and oxidative stress. , Despite extensive research, the availability of effective therapeutic interventions for TBI remains limited. Notably, two phase III clinical trials investigating progesterone as a treatment for TBI failed to demonstrate efficacy, citing poor drug pharmacokinetics as a major challenge. − TBI induces changes in the brain’s extracellular matrix (ECM), which plays a crucial role in modulating cell adhesion, migration, and inflammatory milieu. In particular, reactive astrocytes and microglia express proteoglycans such as tenascin-C in response to injury. , Given the large surface area and injury-responsive nature of the ECM, it represents a compelling target for improving therapeutic delivery and localization. Leveraging the dynamic state of the ECM to guide targeted interventions offers a promising path toward more effective treatments for TBI.

The peptide CAQK (Cys-Ala-Gln-Lys) was identified through in vivo phage display technology in a mouse model of TBI. The putative receptor for CAQK is tenascin-C, which is upregulated after injury. Studies have demonstrated that systemically administered CAQK-targeted nanoscale materials selectively home to injured brain regions. − Moreover, in preclinical TBI models, CAQK exhibited therapeutic benefits by promoting neural repair and functional recovery. However, like many peptide therapeutics, the clinical translation of CAQK is limited due to its short half-life and insufficient accumulation at target sites. − To address the poor pharmacokinetics, nanomaterial-based delivery systems, particularly poly­(ethylene glycol) (PEG) conjugation, have been employed to improve peptide pharmacokinetics by increasing molecular size, enhancing solubility, and shielding nanomaterials from opsonization and phagocytosis. − PEGylation has been widely used in the past decade to enhance drug bioavailability and reduce renal clearance in various FDA-approved nanomedicine applications, including PEGylated liposomes for chemotherapy, PEG-protein conjugates for improved immune response, − and most notably, PEGylated lipid nanoparticles for mRNA delivery to treat COVID-19. − While passive targeting through PEGylation extends systemic circulation, active targeting strategies that employ ligands to engage specific receptors, cells, or proteins have shown enhanced tissue-specific accumulation, particularly within the brain. ,,− In TBI, nanomaterials can exploit BBB dysfunction to facilitate access to injured regions. Therefore, we hypothesized that conjugation of CAQK onto a hydrophilic PEG scaffold (CAQK-PEG) can improve its pharmacokinetics.

We utilized two widely used conjugation strategies to functionalize CAQK onto our polymer carriermaleimide–thiol Michael-type addition and dibenzocylooctyne (DBCO)-azide strain-promoted azide–alkyne cycloaddition (SPAAC). Maleimide functional groups enable the efficient formation of a thiosuccinimide product with the thiol on cysteine residues. This method has been employed in the development of antibody–drug conjugates (ADCs) for targeted cancer therapies such as brentuximab vedotin and trastuzumab emtansine. However, maleimide–thiol linkages are susceptible to hydrolysis and thiol exchange with reactive thiols in albumin and glutathione, which can influence stability, off-target effects, and release kinetics. − Alternatively, SPAAC utilizing DBCO and azide groups offers a bioorthogonal conjugation approach that proceeds rapidly under physiological conditions without the need for a catalyst. This method has been applied in several in vivo drug delivery systems for targeted chemotherapy − and labeling of brain-specific cells and receptors , but is limited in its application toward treating neuroinflammatory diseases due to undesirable biodistribution and immunogenicity. , Recent comparative studies have highlighted the impact of linker chemistry on biodistribution and toxicity, demonstrating that conjugation strategies such as SPAAC can markedly influence immune recognition, clearance kinetics, and therapeutic efficacy. These studies reinforce the need for the careful selection of linkers to align with the intended therapeutic application, ensuring an optimal balance of stability, targeting efficiency, and systemic clearance. Given these considerations, we sought to investigate how these linker chemistries affect the pharmacokinetics of CAQK-PEG following TBI.

We synthesized and characterized CAQK-PEG conjugates using both maleimide–thiol and SPAAC click chemistries with matched peptide stoichiometries across both formulations. Conjugates extended the circulation half-life by ∼90-fold over that of the free CAQK peptide, demonstrating the impact of PEGylation in mitigating rapid peptide clearance. When administered systemically in a mouse model of TBI, we observed widespread distribution of CAQK-PEG conjugates across the perilesional cortex. At the tissue level, we observed that CAQK-PEG was colocalized with tenascin-C, and the CAQK peptide was retained within the injured cortex for 24 h postinjury. While brain accumulation was comparable between the two conjugates, DBCO conjugates exhibited greater off-target retention in filtration organs compared to maleimide conjugates, which displayed more rapid renal clearance. Finally, we hypothesized that because maleimide-linked CAQK conjugates exhibited prolonged circulation time, targeted brain accumulation, and reduced retention in off-target filtration organs, this conjugate could be applied as a prophylaxis to deliver a therapeutic peptide into the injured brain. We achieved 4.5%ID/g CAQK within the injured brain when administered 24 h prior to injury. In summary, we demonstrate the impact of conjugation chemistry of peptide–polymer conjugates in influencing both brain and off-target accumulation, retention, and distribution in a TBI mouse model, emphasizing the role of rational linker selection in the design of peptide-based therapeutics for TBI.

Synthesis of CAQK-Targeted Poly­(ethylene glycol) (PEG) through Two Bioconjugation Strategies

Our goal was to synthesize peptide–polymer conjugates to extend the circulation half-life of a targeting and therapeutic peptide (CAQK) within the injured brain. We selected 40 kDa 8-arm poly­(ethylene glycol) (PEG) as our polymer scaffold for several reasons. First, the terminus of each arm can be readily modified with mono- or bivalent cross-linkers, allowing for multivalent conjugation of targeting moieties. , Second, in previous work, we have established that 40 kDa 8-arm PEG has a ∼10 nm hydrodynamic diameter and passively accumulates in the injured brain following intravenous administration. − Additionally, we have shown that conjugating targeting or therapeutic peptides onto this nanoscaffold can improve the distribution and function of bioresponsive materials within the injured brain. , Finally, PEG is currently applied in multiple FDA-approved nanoformulations to extend the in vivo circulation half-life.

Amine-functionalized 40 kDa 8-arm PEG was batch-reacted with 1 mol equiv of fluorescent molecule AlexaFluor 647 N-hydroxysuccinimide ester (NHS ester) to achieve conjugates with the same labeling ratio, allowing comparative assessment of the in vivo distribution across conjugates based on the fluorescence signal (Figure A, steps 1–2). Following the removal of free unreacted dye, AF-labeled PEG was split into 3 separate reactionsuntargeted mPEG-PEG, CAQK-Mal-PEG, and CAQK-DBCO-PEG. To formulate the untargeted control, AF-labeled PEG was reacted with an excess of methyl-terminated PEG₅-NHS ester to quench the remaining unreacted arms of the nanoscaffold (Figure A, steps 3a–6a). The CAQK peptide used in the synthesis of materials is modified with a fluorescein (FAM) fluorophore on an N-terminal lysine and an azide functional group on the N-terminus of the peptide sequence (azide-FAM-CAQK). We pursued two different conjugation click chemistries to functionalize the peptide on the PEG scaffold. For maleimide-linked conjugates, we used the heterobifunctional cross-linker sulfosuccinimidyl 4-(N-maleimidomethyl)­cyclohexane-1-carboxylate) (Sulfo-SMCC) to cross-link amines on PEG via NHS chemistry and the thiol on the cysteine residue of the peptide via maleimide–thiol Michael-type addition (Figure A, steps 3b–6b). For DBCO-linked conjugates, AF-labeled PEG was modified with the heterobifunctional cross-linker dibenzocyclooctyne-sulfo-N-hydroxysuccinimidyl ester (DBCO-Sulfo-NHS ester) and then covalently clicked to the azide group on the N-terminus of CAQK (Figure A, steps 3c–6c), preserving the thiol on CAQK. By using the same batch of AF-labeled PEG and conjugating the same peptide across the conjugates, we could ensure that any observable differences in the pharmacokinetics of the conjugates were likely due to the conjugation chemistry.

1.

Synthesis, characterization, and purification of CAQK-targeted polyethylene glycol (PEG) nanoscaffold (CAQK-PEG) through two different conjugation strategies. (A) Synthesis scheme of untargeted and targeted PEG conjugates. (B) Absorbance scan of the conjugates following purification of PEG-X-linkers after step 4, normalized to AF647. (C) Absorbance scan of purified CAQK-PEG conjugates after step 6. (D) Polyacrylamide gel comparing mPEG, maleimide, and DBCO-functionalized conjugates during synthesis.

We monitored the progression of the synthesis by measuring the absorbances of the conjugates. The presence of DBCO after step 4 could be measured at 310 nm. , After conjugating and purifying unreacted azide-FAM-CAQK (step 6), we validated that the targeted conjugates have the same relative amount of CAQK per AF647 by measuring the absorbances of FAM and AF647 (Figure C), indicating that we achieved similar CAQK modification. The DBCO peak absorbance at 310 nm decreased as the reaction between DBCO and azide-FAM-CAQK proceeded due to the structural change of a cyclooctyne into a triazole. We measured ∼5 CAQK/PEG for both targeted CAQK-PEG conjugates (Table ). We verified the purification of the conjugates via size exclusion chromatography on a polyacrylamide gel (Figure D). The untargeted and targeted PEG conjugates were assessed for the removal of unreacted AF647 label and FAM-labeled peptides (Table ), with >90% purity of all conjugates. We confirmed peptide functionalization through matrix-assisted laser deposition/ionization time-of-flight (MALDI-TOF) mass spectrometry (Figure S1).

1. Relative Amounts of CAQK/PEG on PEG Conjugates and Purity of Conjugates by the AF647 Fluorophore and Azide-FAM-CAQK Peptide.

conjugate	CAQK/PEG ratio	% purity AF647	% purity FAM-peptide
mPEG	0.0	94	N/A
maleimide	5.2	90	92
DBCO	4.9	96	94

Conjugation of CAQK to Polymer Scaffold Extends Circulation Time and Leads to Distribution in the Injured Brain

To evaluate the impact of PEGylation on CAQK pharmacokinetics, we first measured the circulation half-life of free azide-FAM-CAQK and CAQK-PEG conjugates following systemic injection and a nonlinear 2-phase decay model fit. We calculated a ∼90-fold increase in circulation time for PEG conjugates compared to free peptide (Figure A). The free peptide exhibited rapid clearance with a short blood half-life, consistent with the known kinetics of peptide pharmacokinetics. Peptides have poor in vivo half-life as they can be rapidly excreted into the urine and are susceptible to proteolytic degradation. In contrast, all PEG conjugates demonstrated prolonged circulation, consistent with previous observations that PEG can reduce renal clearance and proteolytic degradation. − Among the PEG conjugates, untargeted PEG, lacking the CAQK targeting moiety, had a similar blood half-life to the targeted conjugates, indicating that systemic clearance dynamics was dominated by PEG and not the peptide.

2.

Pharmacokinetics and biodistribution of CAQK-PEG conjugates in a mouse model of TBI. (A) Blood half-life of free azide-FAM-CAQK and PEG conjugates following retro-orbital injection of 400 nmol PEG/kg (1 mg CAQK/kg); n = 3, mean ± SEM, nonlinear two-phase decay fit. (B) Schematic of the in vivo biodistribution experiment. Representative whole-organ fluorescence surface scans and their quantification measured by AF647-labeled PEG at (C,D) 6 h and (E,F) 24 h post-CCI. Brains were analyzed by integrating the signal from the injured (ipsilateral) vs uninjured (contralateral) hemispheres. PEG surface distributions are compared between materials within each organ (n = 3, mean ± SEM, two-way ANOVA with Tukey’s multiple comparisons test within each organ group, **p < 0.01, ***p < 0.001, ****p < 0.0001).

We then assessed the biodistribution of CAQK-PEG conjugates after systemic delivery in a controlled cortical impact (CCI) mouse model of TBI. CCI is a well-controlled model of TBI that reproducibly elevates molecular phenotypes such as upregulation of tenascin-C. , Mice were injected with 3 mg/kg CAQK, CAQK-PEG, or an equivalent dosage of mPEG-PEG (based on AF647 absorbance) 5 min post-CCI. 6 and 24 h after injection, mice were perfused, and the organs were harvested for surface imaging and histology (Figure B). The ECM target for CAQK, tenascin-C, has been shown to be upregulated at 6 h postinjury. ,,

We performed fluorescence surface scans of the AF647 dye on PEG in whole organs. FAM imaging was not completed due to the poor imaging depth in the tissues. At 6 h postinjury, all PEG conjugates accumulated in the injured brain with signal on par with off-target organs, with preferential localization in the ipsilateral hemisphere over the contralateral hemisphere (Figures C,D and S2). This accumulation of intravenously delivered nanomaterials into the injured hemisphere due to local, transient permeability of the dysregulated BBB is consistent with previous work from our group ,, and others. At 24 h postinjury, more than half of the PEG material is retained in the brain (Figure E,F). Off-target organ analysis revealed distinct distribution patterns between the two conjugation chemistries. DBCO conjugates exhibited significantly higher accumulation than untargeted and maleimide conjugates in filtration organs; DBCO conjugates had greater accumulation in the kidney at both 6 and 24 h and in the liver at 24 h.

Targeted CAQK-PEG Conjugates Accumulate within Injured Brain Tissue and the Peptide Is Retained within the Injured Cortex for 24 h

We further investigated the tissue-level distribution through imaging of coronal brain sections. At 6 h post-CCI, while we did not observe appreciable amounts of free peptide or unmodified PEG (Figure A,B), there was widespread distribution of targeted conjugates irrespective of linkers within the injured cortex based on the fluorescent label of both PEG and peptide (Figure C,D). Interestingly, we observed the PEG signal without peptide in the contralateral hemisphere along the corpus callosum (Figure C,D); the corpus callosum serves as the primary conduit for communication between the left and right hemispheres. While the transport of synthetic materials across the corpus callosum has not been well-documented, demyelination of axons within the corpus callosum after TBI leads to increases in radial diffusivity and isotropic diffusion, which we hypothesize may be linked to the passive diffusion of nanomaterials across the extracellular space.

3.

CAQK-targeted PEG distributes across perilesional cortex in CCI-injured brains and is retained for 24 h after injury. (A–D) Representative coronal brain sections of the whole brain from mice r.o.-administered with free azide-FAM-CAQK, untargeted mPEG, or targeted CAQK-Mal-PEG and CAQK-DBCO-PEG conjugates after 6 and 24 h of circulation (n = 3, blue, nuclei; green, CAQK on conjugates; magenta, AF647 PEG label; scale bar = 1 mm). (E,F) Injured cortex of brains injected with targeted CAQK-PEG conjugates show colocalization with upregulated tenascin-C signal 6 h post-CCI (blue, nuclei; green, CAQK on conjugates; red, tenascin-C; scale bar = 500 μm).

We then confirmed that the localization of CAQK-PEG conjugates coincided with the putative receptor of CAQK, tenascin-C. Immunostaining confirmed that tenascin-C is upregulated only in the injured perilesional tissue, and CAQK-PEG conjugates were enriched in the areas with upregulated tenascin-C (Figure E,F). This observation is consistent with the literature, as other CAQK-targeted nanomaterials have been shown to colocalize with tenascin-C within the injured brain. After 24 h post-CCI, we observed that while there was a loss of signal from the PEG backbone, the CAQK signal was largely retained within the injured cortex (Figure C,D). From surface imaging, we measured similar levels between untargeted and CAQK-targeted PEG (Figure D,F). The discrepancy between surface whole organ imaging and brain slice imaging may be attributed to untargeted PEGylated materials localizing to the injured brain surface without significant penetration into the parenchyma. Studies have shown that imaging depths even with NIR dyes is limited to ∼2–3 mm in the brain, which may over-represent surface signals in a disease such as TBI. , Overall, our results showing increased accumulation of nanomaterials with CAQK modification were consistent with previous studies, which establish that peptide conjugation impacts intratissue distribution of nanomaterials within the injured brain over untargeted controls. ,

Distribution of PEG Conjugates Is Markedly Different in Filtration Organs

To further evaluate the off-target distribution of CAQK-PEG conjugates, we analyzed their accumulation within key filtration organs at 6 and 24 h postinjury (Figure A). We quantified the mean fluorescence intensity (MFI) of fluorescent labels on CAQK (Figure B) and PEG (Figure D) within 5 regions of interest in the liver, kidney, and spleen in triplicate mice. Quantification of MFI revealed that DBCO conjugates exhibited markedly higher retention of peptide and PEG in filtration organs analyzed (liver, kidney, and spleen) compared with maleimide conjugates (Figure B,D). From these images, we also computed Mander’s coefficient to assess the extent of CAQK colocalization with PEG (Figure C) and, vice versa, the extent of PEG colocalization with CAQK (Figure E). We observed that with DBCO conjugates, most of the CAQK remained colocalized with PEG within the liver at 6 h but degraded by 24 h (Figure C). On the contrary, most of the PEG remained colocalized with CAQK within the liver over 24 h, suggesting that while degraded CAQK was removed from organs, DBCO conjugates persisted. Our observation is supported by the hypothesis that the hydrophobic DBCO cross-linker undergoes prolonged retention within the reticuloendothelial system (RES). − Prior studies have shown that hydrophobic materials favor delivery to RES organs, such as the liver, spleen, and kidney, due to adsorption of circulating plasma proteins. − Recently, a study showed that antibody–liposome bioconjugates made via DBCO modification led to 58% higher lung uptake of the nanoparticles, whereas maleimide conjugation resulted in ∼37% higher lung uptake. DBCO conjugation increased complement activation with more contribution via the classical pathway, whereas maleimide conjugation activated complement with more contribution via the alternative pathway, with an overall impact on biodistribution. Although studies that directly compare pharmacokinetics for various linker chemistries are limited, constructs that use DBCO chemistries may require additional design considerations and evaluation relative to those that use maleimide chemistries, as high accumulation in off-target organs can lead to safety and efficacy concerns in later stages of clinical development. Previous studies have also demonstrated that PEGylation alone prolongs circulation time by reducing opsonization and renal clearance. However, our results emphasize that linker chemistry modulates these interactions. We note that in the maleimide-linked conjugation, the peptide is linked through its free sulfhydryl group on the cysteine, and in the DBCO-linked conjugation, the peptide is linked through the N-terminal azide. Previous conjugation with both strategies has described that DBCO-azide chemistry has predefined stoichiometry and a site-directed orientation of the conjugated targeting ligand. The impact of the position of reaction on the peptide is unexplored in our studies, and in particular, free thiols are known to influence pharmacokinetics due to the potential disulfide reaction with carrier proteins. , Furthermore, the lack of a significant untargeted PEG signal in filtration organs suggests that CAQK targeting itself may contribute to increased retention in off-target organs. While we saw similar distribution of maleimide and DBCO-linked conjugates in the perilesional brain cortex, their distribution and elimination kinetics were markedly different in off-target organs, an important consideration for controlling the therapeutic index.

4.

Distribution of CAQK-PEG is markedly different in filtration organs. (A) Representative images of the biodistribution of CAQK-PEG conjugates within the liver, kidney, and spleen at 6 and 24 h post-CCI (blue, nuclei; green, CAQK on conjugates; magenta, AF647 PEG label; scale bar = 200 μm). (B) Change in CAQK mean fluorescence intensity (MFI) in the filtration organs from 6 to 24 h post-CCI across four materials. (C) Change in the proportion of CAQK colocalized with PEG from 6 to 24 h within the liver, kidney, and spleen. (D) Change in PEG MFI in the filtration organs from 6 to 24 h post-CCI across four materials. (E) Change in the proportion of PEG colocalized with CAQK from 6 to 24 h within the liver, kidney, and spleen.

CAQK-PEG as a Peptide Prophylaxis Accumulates in the Injured Brain

There is a high incidence of TBI among military personnel, with over 510,000 service members in the United States alone diagnosed since 2000. Due to the known and elevated risk in this group of individuals, prophylaxis may be a strategy to mitigate negative outcomes. While pharmaceutical interventions, such as antibiotics, steroids, and antiseizure medications, are often used alongside physical protective gear-like helmets and armored vehicles, they largely target mitigating the collateral damage of TBI. Evidence shows that rapid treatment is critical for improving outcomes in TBI, suggesting that prophylactic approaches could significantly mitigate injury severity. We hypothesized that the prolonged circulation time, targeted brain accumulation, and reduced retention in off-target filtration organs of maleimide-linked CAQK-PEG could be leveraged as a prophylaxis to deliver therapeutic peptide into the injured brain. 5 mg/kg CAQK-PEG was r.o.-administered 24 h prior to CCI, and bulk tissue analysis was conducted 6 h post-CCI (Figure A). Quantitative bulk analysis of brain tissue was measured from homogenized organs, and percent injected dose per gram tissue (%ID/g tissue) was calculated based on a standard of known peptide concentrations (Figure B). Even though the material was dosed 24 h prior to injury, we measured 4.5%ID/g CAQK in the injured hemisphere following prophylactic delivery of CAQK-Mal-PEG, whereas free CAQK administration was not detected. Previous studies have observed ∼1%ID/g antibody drug conjugates , or peptide–polymers within healthy or injured brain tissue. Overall, these results support the potential of long-circulating peptide–polymer conjugates to have high bioavailability in a prophylactic dosing regimen.

5.

Prophylactic administration of CAQK-Mal-CAQK 24 h prior to injury can lead to significant accumulation into the injured brain. (A) Schematic of prophylactic administration experiment. (B) Bulk distribution of CAQK-Mal-PEG and free CAQK-FAM within the ipsilateral and contralateral cortices from homogenized tissue (%ID/g, n = 3–4, mean ± SEM, two-way ANOVA with Šídák’s multiple comparisons test, ****p < 0.0001).

Conclusions

CAQK is a targeting peptide identified for its ability to bind ECM components upregulated after traumatic brain injury (TBI), enhancing delivery to injured brain regions. We synthesized and characterized CAQK-PEG conjugates using two widely used linker chemistriesmaleimide–thiol and DBCO-azide. The impact of these chemistries on peptide pharmacokinetics, in particular, off-target organ distribution and retention, was distinct. PEGylation significantly extended the circulation half-life of CAQK compared to that of the free peptide, enhancing accumulation in the injured brain. Both CAQK-PEG conjugates synthesized with either linker effectively distributed to the injured brain and colocalized with tenascin-C in the perilesional cortex; however, maleimide conjugates exhibited lower off-target accumulation in filtration organs. In prophylactic dosing, we show that maleimide-linked CAQK-PEG conjugates significantly outperformed free CAQK peptide in terms of peptide accumulation in the injured brain. Overall, the choice of conjugation chemistry is critical for extending peptide circulation time and minimizing off-target retention in filtration organs, an important consideration for mitigating off-target toxicity and maximizing the therapeutic window. Future studies could explore the use of alternative polymers for conjugation, given the known antibody response to PEG that can accelerate clearance upon repeat administration. Additionally, evaluating linker stability and excretion profiles across off-target organs over time would provide further insight into optimizing linker choice for therapeutic peptide conjugate design.

Experimental Procedures

Synthesis of CAQK-Targeted Poly­(ethylene glycol) (PEG) Conjugates

Azide-FAM-CAQK (azidoacetyl-K­(5FAM)-C6-CAQK) was synthesized by CPC Scientific, Inc. (Sunnyvale, CA). 40 kDa 8-arm PEG amine (hexaglycerol) was purchased from Jenkem Technology (Beijing, China). For the untargeted and targeted conjugates, PEG-amine was first dissolved to 10 g/L in PBS and batch-reacted with 1 mol equiv of AlexaFluor 647-N-hydroxysuccinimide ester (NHS ester) (Thermo) diluted in DMSO. The reaction was stirred at 300 rpm for 30 min at RT, followed by purification on a PD-10 desalting size-exclusion column (Cytiva) into PBS. The reaction was split into three portions to ensure matched AlexaFluor 647 modification for each conjugate. 10 mol equiv of 10 g/L mPEG₅-NHS ester (untargeted mPEG), Sulfo-SMCC (maleimide), or DBCO-Sulfo-NHS ester (DBCO) diluted in DMSO was then added to each corresponding conjugate and stirred at 300 rpm for 1 h at RT. Conjugates were purified with a PD-10 column into PBS and subsequently reacted with 7 mol equiv of azide-FAM-CAQK dissolved in deionized water or an equiv volume of PBS (untargeted PEG). Peptide was reacted overnight at 300 rpm at RT. Following a final PD-10 purification to remove the unreacted peptide, final concentrations were determined by the absorbance of AlexaFluor 647 and FAM using a UV spectrophotometer (Genesys 150). For the prophylaxis in vivo study, 8-arm PEG maleimide (hexaglycerol) (Jenkem Technology; Beijing, China) reacted with 10 mol equiv of CAQK-FAM (K­(5FAM)-C6-CAQK) and quenched with a 20 mol equiv excess of l-cysteine. This conjugate was then dialyzed with PBS, and the concentration of the peptide was measured by the absorbance of FAM. SDS-PAGE was performed with a 12% polyacrylamide gel, AlexaFluor 647 fluorescence from the PEG was imaged on an Odyssey Scanner (Li-Cor Biosciences), and FAM fluorescence from the azide-FAM-CAQK peptide was imaged on a Bio-Rad scanner. MALDI-TOF was performed on a Bruker Autoflex Max. Gaussian filtering was then applied to reduce the noise and facilitate peak detection.

Controlled Cortical Impact (CCI) TBI Mouse Model

Female C57BL/6J mice (8–12 weeks old, Jackson Laboratories) weighing between 15 and 20 g were used for all in vivo studies. Following anesthetization with 2.5% isoflurane, preoperative buprenorphine analgesia was administered. A 5 mm craniotomy was performed over the right hemisphere between the bregma and lambda, and controlled cortical impact was performed using the ImpactOne (Leica Biosystems) with a 2 mm diameter stainless-steel probe at a velocity of 5 m/s, depth of 2 mm, and dwell time of 300 ms. The center of the injury impact was centered around −2.0 mm (±0.5 mm) lateral from the midline and −2.0 mm (±0.5 mm) caudal from the bregma.

Quantification of Conjugate In Vivo Blood Half-Life

Matched doses of 400 nmol PEG/kg or 1 mg CAQK/kg (quantified by AlexaFluor 647 and FAM absorbances) were retro-orbitally injected into mice. 10 μL of blood was collected with an anticoagulant, and fluorescence was measured from the plasma. The %ID of PEG conjugates and peptides was calculated based on a standard of known concentrations and estimated blood volume.

CAQK-PEG In Vivo Study

Five minutes after CCI, 3 mg/kg CAQK (quantified by FAM absorbance) of targeted CAQK-PEG and an equivalent dosage of untargeted mPEG-PEG (quantified by AlexaFluor 647 absorbance) were retro-orbitally injected. Vehicle control received an equivolume of saline. Following 6 and 24 h of circulation time, mice were sacrificed by transcardial perfusion of USP saline, followed by 10% formalin. Each condition was repeated in triplicate for histology.

CAQK-PEG Prophylaxis In Vivo Study

Twenty-four hours before CCI, 5 mg/kg CAQK (quantified by FAM absorbance) was retro-orbitally injected. Vehicle control received an equivolume of saline. Six hours after injury, mice were transcardially perfused with 10 mL of ice-cold PBS for whole-organ homogenization.

Quantitative Biodistribution of Homogenized Tissue

Organ tissue was flash frozen at −80 °C and minced, and lysis buffer (6% w/v sodium dodecyl sulfate (SDS), 150 mM Tris-HCl pH 7.4, 100 mM dithiothreitol (DTT), and 2 mM EDTA) was added to achieve a concentration of 250 mg tissue/mL. Tissue was further processed with a Tissue-Tearor with a 4.5 mm probe (Fisher) at medium-high speed for 20–30 s until the lysate was homogenized. The homogenate was analyzed for FAM fluorescence. The percent injected dose per gram of tissue (%ID/g) was calculated based on a known nanomaterial concentration standard.

Immunostaining of Brain and Organ Tissue Slices

Animals were transcardially perfused with 10% formalin; necropsied organs were further fixed in 10% formalin at 4 °C overnight. Organs were washed in PBS, equilibrated to 30% w/v sucrose–PBS, and frozen in OCT (Tissue-Tek). Coronal brain tissue slices (10 μm thick) were obtained within the 2 mm diameter injury region and then stained using conventional protocols. Briefly, tissues were blocked for 1 h in 2% bovine serum albumin (BSA), 5% serum of secondary antibody, and 0.1% Triton X-100. Primary antibodies (rabbit antifluorescein (Invitrogen) and rat antitenascin C (R&D Systems) were diluted (5 μg/mL and 2.5 μg/gL, respectively) in blocking buffer and incubated with tissue sections overnight at 4 °C. Secondary antibody staining and counterstaining with Hoechst were done following standard protocols, and slides were mounted with Fluoromount-G (Southern Biotech). Images were collected on a Nikon Eclipse Ti2 microscope fitted with a Hamamatsu Orca-Flash 4.0 digital camera. Images for direct comparison were collected using the same exposure and LED intensity settings. For organ tissue imaging, images were acquired by a researcher blinded to the conditions. Measurements were made from 5 regions of interest identified from Hoechst imaging per triplicate organ.

Software and Statistics

GraphPad Prism (10.4.1) was used to perform the statistics. All images were processed in ImageJ (2.16). Mander’s coefficients were generated using the BIOP JACOP ImageJ plugin. Synthesis schematic was generated using ChemDraw (22.0). Abstract schematic was created with BiorRender.com

Safety Statement

All experiments involving hazardous chemicals were carried out following institutional guidelines, with appropriate protective measures, as outlined in the associated MSDS sheets.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.bioconjchem.5c00175.

• MALDI-TOF results and triplicate NIR organ scans (PDF)

The authors declare no competing financial interest.

Published as part of Bioconjugate Chemistry special issue “Brain-Targeted Drug Delivery”.