In vivo metabolic tagging and targeting of circulating red blood cells

Domain: biology
Published: 2026-03-21
Editorial outcome: major_revision

Abstract
Engineering red blood cells (RBCs) has been widely explored for drug delivery, imaging, vaccination, and other applications. However, effective strategies to directly engineer RBCs in vivo are still lacking. Here we report successful metabolic glycan labeling of RBCs in vivo. We demonstrate that systemically administered azido-sugars can metabolically label circulating RBCs with azido groups, through labeling of both mature RBCs and RBC precursor cells. The surface azido tags on RBCs can persist for over 42 days in female mice (nearly the lifespan of RBCs), while tags on leukocytes decay to negligible levels within 3 days. Azido-labeled RBCs can covalently capture dibenzocyclooctyne-bearing cargos in vivo via click chemistry, extending cargo circulation from hours to over 35 days. This RBC tagging and targeting technology can improve fluorescence imaging of blood vessels, enable long-term MRI of brain vasculatures with a single gadolinium dose, and improve the pharmacokinetics of drugs.

Introduction
Red blood cells (RBCs) constitute 40-45% of whole blood volume and >99% of blood cells, and are responsible for transporting oxygen to all tissues.1,2 RBC dysfunction is often associated with severe diseases and life-threatening complications, demanding compatible and effective tools to probe and modify aberrant RBCs.3-5 Considering their long life span (~120 days in human and ~45 days in mice) and ability to access deep tissues,6,7 RBCs are also an attractive engineering target for drug delivery, imaging, hemostasis, vaccination, and other applications.8-13 For example, drugs bounded to RBCs could exhibit improved pharmacokinetics and tissue accumulation, potentially achieving better therapeutic efficacy with a reduced dose of drugs.14-16 Contrast agents conjugated to circulating RBCs also enable long-term imaging of vasculatures and tissues. However, a safe and effective method to directly modify or engineer circulating RBCs in vivo still does not exist. Existing RBC engineering approaches mostly rely on isolating and modulating RBCs under ex vivo conditions, and the whole process of isolating RBCs, modifying RBCs ex vivo, and infusing engineered RBCs is time-consuming and costly, could cause damage to RBCs, and suffers from a risk of infections.9,17-19 Significant progress has indeed been made to address these issues and bring RBC-based drug delivery systems to clinical trials.20 In parallel, we envision direct in vivo engineering of RBCs will provide possibilities for better understanding of RBC biology and developing enhanced diagnostics and therapies for a variety of diseases.

Modification of RBCs was attempted via non-covalent or covalent methods. Non-covalent methods involve physical adsorption of cargos onto RBC membranes via electrostatic interactions11,21,22 or binding of a targeting ligand to RBC receptors (e.g., Ter119).13,23-25 The former is limited by the weak interactions between cargos and RBCs and the need of custom-designed adsorbable structures,11,21,22 while the latter is limited by the lack of specificity and low abundance of RBC receptors.13,23-25 The covalent conjugation of cargos to RBCs via amine-carboxyl chemistry was also attempted, but the density of amine groups on RBC membranes is minimal and the modified RBCs often exhibit impaired function and viability.26,27 Another covalent method involves the genetic engineering of cell-surface proteins with an LPXTG motif, followed by the conjugation of (Gly) -bearing molecules in the presence of sortase.24,28,29 However, the necessity of introducing the sortase-cleavable LPXTG motif via gene transduction poses a notable drawback, especially for in vivo applications. To date, a facile, safe, and universal method for in vivo engineering and cargo attachment of RBCs still does not exist.

Here we report a facile and safe strategy to metabolically label RBCs with unique chemical tags (e.g., azido groups) in vivo (Fig. 1a). We show that unnatural sugars such as tetraacetyl-N-azidoacetylmannosamine (AAM), upon systemic administrations, can metabolically label RBCs with azido groups in the form of glycoproteins and glycolipids via the metabolic glycoengineering processes.30-33 The type, dose, and dosing frequency of azido-sugars all impact the overall RBC labeling efficiency. It is noteworthy that non-specific azido-sugars used in this study can label both RBCs and non-RBCs in vivo. However, due to the non-proliferative property and long life-span of RBCs, the installed azido groups are stable in the bloodstream for over 42 days, nearly the life-span (~45 days) of mouse RBCs. In contrast, the azido tags on the membrane of leukocytes in the blood and cells in healthy tissues such as liver, spleen, lung, heart, brain, and kidney decay to baseline levels within 3 days (Fig. 1a). We further demonstrate that the azido-labeled RBCs can rapidly capture dibenzocyclooctyne (DBCO)-bearing cargos in the bloodstream via efficient click chemistry.34-38 Strikingly, the conjugated cargos can retain on the surface of RBCs for over 35 days in mice (Fig. 1a), without causing any noticeable acute or chronic toxicity. This could be attributed to the unique physiology of RBCs and opens up opportunities for various diagnostic and therapeutic applications. As a few examples, we demonstrate that the RBC labeling and targeting technology enables in vivo conjugation of fluorophores onto circulating RBCs for enhanced fluorescence imaging of vasculatures and tumors (Fig. 1b), conjugation of gadolinium (Gd) onto RBCs for long-term magnetic resonance imaging (MRI) of brain blood vessels (for over 11 days with a single dose of Gd) (Fig. 1c), and conjugation of insulin and other drugs for enhanced pharmacokinetics and therapeutic efficacy (Fig. 1d).

Results
In vitro metabolic labeling of RBCs
We first screened the metabolic labeling efficiency of three types of azido-sugars, AAM, tetraacetyl-N-azidoacetylgalactosamine (AAGal), and tetraacetyl-N-azidoacetylglucosamine (AAGlu) using murine erythroleukemia (MEL) cells, a precursor cell of RBCs. MEL cells were incubated with 50 µM AAM, AAGal, or AAGlu for varied times, followed by the detection of cell-surface azido groups using DBCO-Cy5. Compared to control cells without azido-sugar treatment, cells treated with AAM, AAGal, or AAGlu showed significantly higher Cy5 fluorescence intensity (Supplementary Fig. 1a-c), demonstrating the successful labeling of MEL cells with azido groups. AAM exhibited a higher labeling efficiency than AAGal and AAGlu (Supplementary Fig. 1a-b). As MEL cells can proliferate rapidly, the amount of cell-surface azido groups decreased from 24 h to 48 h (Supplementary Fig. 1d-f). By adding fresh AAM to the media at 48 h, the density of cell-surface azido groups could be further increased (Supplementary Fig. 1d-f). It is noteworthy that AAM treatment induced negligible changes in the viability and differentiation of MEL cells (Supplementary Fig. 1g-h).

We next studied whether AAM can metabolically label primary RBCs in vitro (Supplementary Fig. 2a). As conventional Alsever’s solution and FBS-containing DMEM media failed to maintain the long-term survival of primary RBCs, we tested different combinations of medium components and narrowed down to a type of DMEM medium containing 10% FBS, 5% BSA, non-essential amino acids, and 10 g/L glucose, which was able to maintain the high viability of RBCs isolated from C57BL/6 mice for up to 120 h in vitro. Mouse RBCs were incubated with AAM for 24 h, and the cell-surface azido groups were detected by DBCO-Cy5. Compared to control cells, AAM-treated RBCs showed significantly higher Cy5 fluorescence intensity (Supplementary Fig. 2b-c), indicating the successful labeling of RBCs with azido groups. We also studied whether AAM treatment could induce the apoptosis of RBCs by examining the expression level of phosphatidylserine, which showed negligible differences between AAM- and PBS-treated RBCs (Supplementary Fig. 2d-e). These experiments demonstrated that AAM can metabolically label primary RBCs with azido groups in vitro, without inducing the apoptosis or death of RBCs.

In vivo metabolic labeling of RBCs
We next studied whether systemically administered AAM can metabolically label RBCs with azido groups in vivo (Fig. 2a). C57BL/6 mice were intravenously administered with AAM (100 mg/kg) twice daily for three days, followed by the collection of RBCs at different times for azido detection via DBCO-Cy5. Compared to control RBCs that showed minimal Cy5 signal, RBCs collected from AAM-treated mice at 14 days post AAM injections showed a distinct Cy5+ population in FACS assay (Fig. 2b), indicating the successful metabolic labeling of RBCs with azido groups. Fluorescence imaging also confirmed the much higher Cy5 fluorescence intensity of RBCs from AAM-treated mice (Fig. 2c-d). The percentage of azide+ RBCs increased from 4.2% at 24 h to 10.4% at 5 days, and further to 11.3% at 7 days (Fig. 2e). While the percentage of azide+ RBCs started to decrease from day 7, a notable fraction of azide+ RBCs could be consistently detected for over 42 days (Fig. 2e-f), nearly the life-span of mouse RBCs (~45 days). The Cy5 fluorescence intensity ratio of RBCs (AAM/PBS) constantly stayed above 1.0, with a maximal ratio of 3.1 at 7 days (Fig. 2g). These experiments demonstrated the successful metabolic labeling of RBCs with azido groups in vivo and the superior retention of azido groups on the surface of RBCs. It was not surprising to detect azido-labeled immune cells and other cells in the bloodstream at 24 h post the injections of AAM (Fig. 2h-i). However, due to their high proliferation rate and active metabolisms, the number of azido groups decayed to the baseline level after 3 days (Fig. 2h-i). The number of azide+ RBCs is 28-fold, 93-fold, 583-fold, and 3,844-fold of azido+ white blood cells (WBCs) at 0.5, 2.5, 4.5, and 7.5 days post the injections of AAM (Fig. 2j-k, Supplementary Fig. 3a). It is noteworthy that AAM treatment did not alter the percentage and number of RBCs and WBCs in the bloodstream (Supplementary Fig. 3b).

To further confirm the successful metabolic labeling of RBCs, we performed magnetic selection of azido-positive RBCs collected from C57BL/6 mice at 7 days post injections of AAM via DBCO-desthiobiotin conjugation, streptavidin-microbead pull-down, and biotin elution (Fig. 2l). Flow cytometry analyses confirmed the successful enrichment of desthiobiotin-tagged RBCs (Fig. 2m-n, Supplementary Fig. 3c). Western blot analysis of the enriched azido/desthiobiotin-tagged RBCs versus control azido- RBCs was able to identify several types of azido-labeled proteins such as band 3 proteins (100 kDa), bands 4 and 5 proteins (55-60 kDa), and glycophorins A–D (30-35 kDa) (Fig. 2o). It is noteworthy that western blot analysis of RBCs, without magnetic enrichment, also showed a difference between AAM and PBS groups (Supplementary Fig. 3d-e).

Mechanisms underlying in vivo metabolic labeling of RBCs
We further studied whether azido-labeled glycoproteins are primarily expressed on RBC membrane, by conjugating RBCs from AAM-treated mice with DBCO-desthiobiotin, isolating the membrane and cytoplasmic proteins, and performing western blot analysis (Fig. 3a). The cytoplasmic proteins of RBCs in both AAM and PBS groups showed minimal desthiobiotin signal (Fig. 3b). In comparison, the membrane proteins of RBCs in AAM group showed significantly higher desthiobiotin signals (Fig. 3b). These results demonstrated that azido-labeled glycoproteins mainly exist on the outer membrane instead of in the cytoplasm. To further validate the presence of azido-labeled glycoproteins, the membrane proteins of desthiobiotin-conjugated RBCs were treated with PNGase F, an enzyme catalyzing the removal of N-linked oligosaccharide chains from glycoproteins, for 1 h prior to western blot analysis. As a result, PNGase F treatment eliminated nearly all the desthiobiotin signal from proteins (Fig. 3c), validating the metabolic expression of azido groups in the form of glycoproteins in azido-labeled RBCs. We also tested the presence of azido-tagged glycolipids on the membrane of RBCs isolated from AAM-treated mice, by incubating RBCs with DBCO-Cy5, extracting membrane lipids from RBCs, and analyzed with HPLC under the fluorescence mode. Lipids isolated from AAM-treated 4T1 cancer cells were used as the positive control. As a result, HPLC analyses confirmed the presence of Cy5-tagged lipids in AAM-treated RBCs and 4T1 cells instead of PBS-treated RBCs (Fig. 3d, Supplementary Fig. 3f).

We next studied whether metabolic labeling of RBC precursor cells in the bone marrow contributes to the overall RBC labeling efficiency. Mouse bone marrow cells were harvested at 48 h after AAM injections and stained with DBCO-Cy5 to analyze azido-labeled RBC precursor cells including proerythroblasts, basophilic erythroblasts, polychromatophilic erythroblasts, orthochromatophilic erythroblasts, and reticulocytes (Supplementary Fig. 4a). Compared to the PBS group, basophilic erythroblasts, polychromatophilic erythroblasts, orthochromatophilic erythroblasts, and reticulocytes in the AAM group showed a higher Cy5 fluorescence intensity (Fig. 3e). The ratio of Cy5 mean fluorescence intensity (AAM/PBS) increased with the stage of erythropoiesis (Fig. 3f). These results demonstrate that systemically administered AAM can metabolically label RBC precursor cells in the bone marrow, which could contribute to the overall RBC labeling efficiency. It is noteworthy that AAM treatment did not alter the percentage and number of erythroid precursor cells (Supplementary Fig. 4b-c).

We also assessed and compared in vivo RBC labeling efficiency of AAM, AAGal, and AAGlu. C57BL/6 mice were intravenously injected with AAM, AAGal, AAGlu, or PBS twice daily for three days, and RBCs were collected at different times and stained with DBCO-Cy5 for flow cytometry analysis. Compared to AAGlu, AAM and AAGal showed a significantly higher RBC labeling efficiency (Fig. 3g-h). Compared to the AAGal group, azido-labeled RBCs in AAM-treated mice exhibited a higher stability over time (Fig. 3g-h). In addition to intravenous injections, we showed that intraperitoneal injections of AAM, which allowed for the use of a higher dose, also resulted in successful metabolic labeling of RBCs with azido groups (Supplementary Fig. 5a-d). The RBC metabolic labeling efficiency increased with the dosing frequency of AAM, with up to 14.5% of azido-labeled RBCs among all the circulating RBCs (Supplementary Fig. 5a-d).

In vivo metabolic labeling of RBCs does not induce noticeable toxicity
We also studied whether in vivo metabolic labeling of RBCs could pose any toxic effect on blood cells and tissues. RBCs from AAM- or PBS-treated mice both showed the normal biconcave disc morphology and exhibited minimal agglutination (Fig. 4a), which was further confirmed by flow cytometry (Fig. 4b). As a key indicator of RBC metabolic states, the intracellular ATP level was measured and compared between AAM and PBS groups. Per the ATP bioluminescence assay, RBCs from AAM- or PBS-treated mice showed negligible differences in intracellular ATP levels (Fig. 4c). In addition to ATP, we also measured the intracellular NAD(H)/NADP(H) levels of RBCs using the resazurin assay. Compared to control RBCs, RBCs in AAM-treated mice showed negligible changes in NAD(H)/NADP(H) levels, both showing a much higher NAD(H)/NADP(H) level than the negative control (Fig. 4d). The RBC and WBC counts showed no differences between the AAM and PBS groups (Fig. 4e-f). Different subtypes of WBCs, including T cells, B cells, and granulocytes, also showed no differences between AAM and PBS groups (Fig. 4g). To study the potential chronic toxicity, organs were harvested at 6 weeks post AAM injections, sectioned, and stained with H&E. Compared to the PBS group, AAM treatment did not induce any noticeable toxicity against all the examined tissues including liver, spleen, kidney, heart, and lung (Fig. 4h). We also analyzed the potential metabolic labeling of cells in healthy tissues. At 4 days post injections of AAM or PBS, tissues were homogenized and the extracted proteins were incubated with DBCO-biotin prior to western blot analysis. Tissues including brain, liver, spleen, kidney, heart, and lung showed negligible differences between AAM and PBS groups (Fig. 4i). Confocal imaging of tissue sections, after staining with DBCO-Cy5, also showed negligible differences in Cy5 fluorescence intensity between AAM and PBS groups (Fig. 4j-k). We also analyzed the membrane osmotic and mechanical fragility of RBCs collected from AAM-treated mice (both azide-positive and azide-negative fractions) and PBS-treated mice, which showed negligible difference (Supplementary Fig. 6a-b). RBC indices including RBC counts, Hemoglobin concentration, hematocrit percentages, mean cell volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), and red cell distribution width (RDW) (Supplementary Fig. 7a-g), as well as RBC morphology (Supplementary Fig. 7h), showed negligible differences between AAM and PBS groups. These data demonstrated that AAM-mediated metabolic labeling induced negligible changes in RBC membrane fragility and RBC functions.

In vivo targeting of DBCO-cargo to azido-labeled RBCs
We next studied whether circulating azido-labeled RBCs would enable targeted conjugation of DBCO-molecules via click chemistry in vivo. C57BL/6 mice were intravenously injected with AAM or PBS twice daily for three days, followed by intravenous injection of DBCO-Cy5 at three days post the last injection of AAM (Fig. 5a). At 3 h post injection of DBCO-Cy5, 3.7% of RBCs in AAM-treated mice were Cy5-positive, which was much higher than 0.8% in PBS-treated mice (Fig. 5b). Mean Cy5 fluorescence intensity of RBCs from AAM-treated mice was also significantly higher than control RBCs (Fig. 5c-d). At 9 h post injection of DBCO-Cy5, the percentage of Cy5+ RBCs and mean Cy5 fluorescence intensity stayed much higher in the AAM-treated mice than in the control mice (Fig. 5b-d). After 24 or 48 h when the vast majority of the injected DBCO-Cy5 should have been cleared from the bloodstream, a significantly higher number of Cy5+ RBCs were still detected in AAM-treated mice than in control mice (Fig. 5b-d). To monitor the long-term retention of Cy5+ RBCs in the bloodstream, we also harvested RBCs at 5, 7, 14, 21, 28, and 35 days, respectively post the injection of DBCO-Cy5, which all showed higher numbers of Cy5+ RBCs in AAM-treated mice than in control mice (Fig. 5b-d). As expected, blood isolated from AAM-treated mice at days 1, 4, 7, and 14 also showed higher Cy5 fluorescence intensity than blood from PBS-treated mice (Fig. 5e-f). Among DNA-containing cells (non-RBCs) in the bloodstream, the accumulation of DBCO-Cy5 showed negligible differences between AAM and PBS groups at all examined times (Fig. 5g-h), demonstrating minimal off-target delivery of DBCO-Cy5 to non-RBC cells. This is consistent with the labeling results, which showed that the percentage of azide+ cells among DNA-containing blood cells decayed to baseline levels at 3 days post AAM injection (Fig. 2h-k). We also examined the potential off-target accumulation of DBCO-Cy5 in healthy tissues. At 24 h post DBCO-Cy5 injection, confocal imaging of tissue sections showed negligible differences in Cy5 fluorescence intensity between AAM and PBS groups (Fig. 5i-j). To better understand the membrane retention of conjugated DBCO-Cy5 on RBCs, we also isolated the RBCs at 7 days post injections of AAM, incubated them with DBCO-Cy5, and monitored the membrane retention of Cy5 ex vivo (Supplementary Fig. 8a). As expected, at 1, 3, and 5 days post DBCO-Cy5 conjugation, RBCs from AAM-treated mice consistently showed much higher Cy5 fluorescence intensity than RBCs from PBS-treated mice (Supplementary Fig. 8b-c), substantiating the excellent membrane retention of Cy5 conjugated onto RBCs. These experiments demonstrated that azido-labeled circulating RBCs can covalently capture DBCO-molecules in vivo and the conjugated molecules can retain on RBC membrane for >5 weeks in mice. We are aware of the concern on the potential immunogenicity of cargos being conjugated to circulating RBCs. In separate studies, we attempted to conjugate DBCO-functionalized 2,4-dinitrophenylated Hemocyanin, Keyhole Limpet (DNP-KLH) and DBCO-ovalbumin (OVA), two representative antigens, to RBCs in vivo and monitor the antibody titers over time. As a result, the in vivo conjugation of DNP-KLH and OVA antigens failed to exert any enhancement on the antibody responses compared to the control groups (Supplementary Fig. 9), which lessens the concern of potent immunogenicity of RBC-conjugated cargos in our metabolic labeling approach. We estimate <1-10% of administered DBCO-protein could be conjugated to circulating RBCs, and the in vivo RBC labeling and targeting conditions require further optimization to increase the amount of conjugated cargos.

Long-term fluorescence imaging of blood vessels and tumors
Considering that DBCO-Cy5 can be conjugated to azido-labeled RBCs and well retain on RBCs in vivo, we next explored its promise for long-term fluorescence imaging of blood vessels and tissues. Balb/c mice were intravenously injected with AAM or PBS twice daily for three days, followed by the subcutaneous inoculation of 4T1 tumor cells and intraperitoneal injection of DBCO-Cy5 at 4 and 7 days, respectively (Fig. 6a). Starting from 1 h post injection of DBCO-Cy5, a higher Cy5 fluorescence intensity was detected in RBCs from AAM-treated mice than RBCs from PBS-treated mice (Fig. 6b). IVIS imaging also showed a higher Cy5 fluorescence intensity of 4T1 tumors in AAM-treated mice than in PBS-treated mice at 21 days post DBCO-Cy5 injection (Fig. 6c-d). Ex vivo imaging of 4T1 tumors confirmed a higher Cy5 fluorescence signal in the AAM group (Fig. 6e-f). Indeed, blood vessels in 4T1 tumors of AAM-treated mice could be clearly visualized (Fig. 6e). Similarly, the blood vessels in other tissues, including the terminal arterioles structures in the spleen, could be visualized (Supplementary Fig. 10a). In another setting, C57BL/6 mice were intravenously injected with AAM or PBS twice daily for three days, followed by intravenous injection of DBCO-Cy5 on day 0 and B16F10 tumor inoculation on day 17 (Supplementary Fig. 10b). On day 24, i.e., 7 days post the subcutaneous inoculation of B16F10 cells, B16F10 tumors from AAM-treated mice showed a 1.92-fold Cy5 fluorescence intensity in comparison with tumors from PBS-treated mice (Supplementary Fig. 10c-d), presumably due to the presence of Cy5-conjugated RBCs within the tumor vasculatures. Consistently, we observed a higher Cy5 fluorescence signal in the liver, kidney, and other organs of AAM-treated mice than those of PBS-treated mice (Supplementary Fig. 10e-f).

MRI imaging of brain blood vessels
As cargos can be conjugated to circulating RBCs and retain on RBCs for weeks via the RBC labeling and targeting technology, we next investigated whether MRI contrast agents (e.g., Gd) can be tagged onto RBCs in vivo for long-term MRI, using contrast-enhanced (CE) imaging of brain blood vessels as an example. We first synthesized DBCO-DOTA-Gd, by reacting DBCO-NH with DOTA-COOH to yield DBCO-DOTA and furthering complexing DBCO-DOTA and Gd3+ (Supplementary Fig. 11a). The chemical structure of DBCO-DOTA and DBCO-DOTA-Gd was characterized by 1H NMR spectroscopy (Supplementary Fig. 11b), HPLC (Supplementary Fig. 11c), and mass spectrometry (Supplementary Fig. 11d). For the in vivo MRI study, C57BL/6 mice were intravenously injected with AAM or PBS twice daily for three days, followed by intravenous injection of DBCO-DOTA-Gd after 4 days (Fig. 6g). Before the injection of DBCO-DOTA-Gd, the blood vessels were barely visible in a CE T1-weighted MR angiography scan (CE-MRA), with or without maximum intensity projection (MIP) (Fig. 6h). At 10 min post the injection of DBCO-DOTA-Gd, blood vessels were clearly visible in both AAM- and PBS-treated mice using the same CE-MRA sequence (Fig. 6h). At 24 h when free DBCO-DOTA-Gd was largely cleared, while MIP could visualize the main blood vessels in both AAM and PBS group, only the AAM group showed visible branched vessels (Fig. 6h), showing excellent retention of DBCO-DOTA-Gd on the azido-labeled RBCs. Without RBC labeling, Gd would need to be injected again for CE-MRA scans, as demonstrated in the PBS group. Strikingly, this signal enhancement was still detected even at 4 or 11 days post injection of DBCO-DOTA-Gd for the AAM group (Fig. 6h). To further compare the signals, we analyzed two specific coronal views of the raw MR images for both day 4 and day 11. Compared to PBS-treated mice, AAM-treated mice showed enhanced MR signals in several blood vessels on both day 4 and day 11 (Fig. 6i, 6k). Quantification of the signals indicated a 1.23-fold enhancement for AAM group on day 4 and a 1.13-fold enhancement on day 11 (Fig. 6j), which was consistent in different coronal views (Fig. 6j, 6l). These experiments demonstrated the feasibility of conjugating Gd to circulating RBCs for long-term MRI of blood vessels.

In vivo conjugation of drugs to RBCs for improved pharmacokinetics and efficacy
In addition to imaging agents, we envision the RBC labeling and targeting approach can be utilized to improve the pharmacokinetics of therapeutics. The feasibility of conjugating small-molecule doxorubicin and macromolecular phycoerythrin (PE) onto azido-labeled RBCs was first tested. DBCO-doxorubicin with a pH-labile hydrazone linkage was synthesized (Supplementary Fig. 12a-b). Following intravenous injection of AAM or PBS into C57BL/6 mice twice daily for three days, DBCO-doxorubicin was intravenously administered (Supplementary Fig. 12c). At 2 h post the injection of DBCO-doxorubicin, a higher fraction of doxorubicin-containing RBCs was detected in AAM-treated mice than in control mice (Supplementary Fig. 12d). Similarly, PE, upon DBCO functionalization (Supplementary Fig. 12e), can conjugate to azido-labeled RBCs via click chemistry (Supplementary Fig. 12f-h). After demonstrating the feasibility of conjugating different cargos to RBCs, we next explored whether DBCO-insulin can be conjugated to circulating RBCs for improved pharmacokinetics and therapeutic efficacy. DBCO-insulin with a hydrolysable ester linkage was synthesized and characterized (Fig. 7a, Supplementary Fig. 13a-b). By incubating AAM- or PBS-treated 4T1 cells with DBCO-insulin and detecting the cell-surface insulin using rabbit anti-insulin and Cy5-conjugated goat anti-rabbit secondary antibody, a higher Cy5 fluorescence signal was detected in AAM-treated 4T1 cells than in control cells (Supplementary Fig. 13c-d), demonstrating the successful conjugation of DBCO-insulin. DBCO-insulin was also able to conjugate to azido-labeled RBCs that were isolated from AAM-treated mice, as evidenced by antibody staining (Fig. 7b, Supplementary Fig. 13e). In vivo, following the intravenous injection of AAM or PBS into streptozotocin (STZ)-pretreated mice,39,40 DBCO-insulin or unmodified insulin was intraperitoneally injected. At 8 h post the injection, a significantly higher level of insulin was detected in the plasma of mice injected with AAM and DBCO-insulin than the control mice (Fig. 7c-d), demonstrating the successful conjugation of DBCO-insulin to azido-labeled RBCs in vivo and subsequent release of insulin from RBCs. We have also monitored the blood concentration of insulin over an extended time. Mice were treated with AAM to enable in vivo RBC labeling, followed by i.p. injection of DBCO-ester-insulin. The blood concentration of insulin was measured via ELISA. For all the selected time points between 1 and 24 h post DBCO-ester-insulin injection, AAM-treated mice exhibited a higher plasma insulin level than PBS-treated mice (Supplementary Fig. 13f), supporting the prolonged blood circulation of drugs via our RBC labeling and targeting approach. However, it is noteworthy that the ester linkage between DBCO and insulin is easily degradable under in vivo conditions, and we envision the tunability of drug release kinetics with the use of different chemical linkages. In the subsequent glucose tolerance test, AAM-mediated RBC labeling combined with DBCO-insulin improved the control of blood glucose levels, in comparison with the non-targeting groups (Fig. 7e-f, Supplementary Fig. 13g). In consistence with the glucose control result, diabetic mice treated with AAM and DBCO-insulin showed the fastest recovery in body weight than other groups (Fig. 7g-h). These experiments demonstrated the promise of the RBC labeling and targeting technology to enhance the pharmacokinetics and therapeutic efficacy of drugs.

Methods
Ethical statement. This research complies with all relevant ethical regulations. All procedures involving animals were done in compliance with National Institutes of Health and Institutional guidelines with approval from the Institutional Animal Care and Use Committee at the University of Illinois at Urbana-Champaign (Protocol #24177).

Materials and Instrumentation. Mannosamine hydrochloride, galactosamine hydrochloride, glucosamine hydrochloride, dicyclohexyl carbodiimide, N-hydroxysuccinimide, and other chemical reagents were purchased from Sigma Aldrich (St. Louis, MO, USA) unless otherwise noted. Primary antibodies used in this study were obtained from Thermo Fisher Scientific (Waltham, MA, USA). All antibodies were diluted according to the manufacturer’s recommendations. Fixable viability dye efluor780 was obtained from Thermo Fisher Scientific (Waltham, MA, USA). SYTO™ 85 Orange Fluorescent Nucleic Acid Stain was purchased from Thermo Fisher Scientific (Waltham, MA, USA). ENLITEN® ATP Assay System Bioluminescence Detection Kit was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Histopaque®-1077 was purchased from Sigma Aldrich (St. Louis, MO, USA). Halt™ Protease Inhibitor Cocktail was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Nitrocellulose membrane was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Insulin ELISA kit (Catalog 80-INSHU-E01.1) was purchased from ALPCO (Salem, NH, USA). Clarity BG1000 Blood Glucose Meter and Strips were purchased from VWR International, LLC (Radnor, PA, USA). FACS analyses were collected on Attune NxT or BD LSR Fortessa flow cytometers and analyzed on FlowJo v7.6 and FCS Express v6 and v7. Fluorescent images were taken with a EVOS microscope (Thermofisher, Waltham, MA, USA). Confocal laser scanning microscopy (CLSM) images were taken by using a Zeiss LSM 700 Confocal Microscope (Carl Zeiss, Thornwood, NY, USA). High-performance liquid chromatography (HPLC) analysis was performed on a Shimadzu CBM-20A system (Shimadzu, Kyoto, Japan) equipped with an SPD-20A PDA detector (190–800 nm), an RF-20A fluorescence detector, and an analytical C18 column (Shimadzu, 3 μm, 50 × 4.6 mm, Kyoto, Japan). Nuclear Magnetic Resonance (NMR) spectra were recorded on a Varian U500 (500 MHz) or VXR500 (500 MHz), or a Bruker Carver B500 (500 MHz) spectrometer. Electrospray ionization (ESI) mass spectra were obtained from a Waters ZMD Quadrupole Instrument (Waters, Milford, MA, USA). Matrix-assisted laser desorption/ionization (MALDI) spectra were collected on the Bruker Ultraflextreme MALDI-TOF/TOF Mass Spectrometer. In vivo and ex vivo images of animals and tissues were taken on a Bruker In Vivo Imaging System (Bruker, Billerica, MA, USA). Preparative HPLC was performed on a CombiFlash®Rf system (Teledyne ISCO, Lincoln, NE, USA) equipped with a RediSep®Rf HP C18 column (Teledyne ISCO, 30 g, Lincoln, NE, USA). Lyophilization was conducted in a Labconco FreeZone lyophilizer (Kansas City, MO, USA). Magnetic resonance imaging (MRI) scans were performed on a 9.4 T Bruker 30 cm AVANCE NEO equipped with a dual channel transmit, 4 receiver channels, a 4-channel mouse brain array, and a 4-channel rat brain array coil. The following fluorophore-conjugated anti-mouse antibodies were used: PE-anti-CD45 (clone 30-F11; Thermo Fisher Scientific, cat. no. 12-0451-82; dilution 1: 200), PE-Cy7-anti-CD44 (clone IM7; Thermo Fisher Scientific, cat. no. 25-0441-82; dilution 1:200), and FITC-anti-TER119 (clone TER-119; Thermo Fisher Scientific, cat. no. 11-5921-82; dilution 1:200). Phosphatidylserine staining was performed using Annexin V-FITC (Thermo Fisher Scientific, cat. no. A13199; dilution 1:200). All antibodies and reagents were validated by the manufacturer for flow cytometry applications.

Cell lines and animals. The MEL cell line was a generous gift from Dr. Martin Burke’s lab at the University of Illinois at Urbana-Champaign. The murine breast carcinoma cell line 4T1 (female, BALB/c origin; ATCC® CRL-2539™) and the murine melanoma cell line B16-F10 (male, C57BL/6 origin; ATCC® CRL-6475™) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). MEL cells were cultured in DMEM containing 10% FBS, 100 units/mL Penicillin G, non-essential amino acids, and 5% BSA at 37oC in 5% CO2 humidified air. 4T1 and B16F10 cells were cultured in DMEM containing 10% FBS, 100 units/mL Penicillin G at 37oC in 5% CO2 humidified air. Cell lines were used at low passage after thawing and were routinely tested for mycoplasma contamination using a commercial detection kit and were confirmed negative prior to experiments. Female C57BL/6 and Balb/c mice (6-8 weeks) were purchased from the Jackson Laboratory (Bar Harbor, ME, USA). Only Female mice were used in this study, due to the challenge in housing a large number of male mice. Feed and water were available ad libitum. Artificial light was provided in a 12 h/12 h cycle.

Synthesis of AAM, AAGal and AAGlu. Mannosamine hydrochloride or galactosamine hydrochloride or glucosamine hydrochloride (1.0 e.q.) was dissolved in anhydrous methanol and cooled to 0oC, followed by the addition of sodium methoxide in methanol (1.0 e.q.). Chloroacetic anhydride (1.05 e.q.) and triethylamine (1.0 e.q.) were then added. The reaction mixture was stirred at room temperature for overnight. Upon removal of the solvent, the crude product was re-dissolved in water. Sodium azide (4.0 e.q.) was then added and the reaction mixture was stirred at 60℃ for overnight. After removing the solvent, the crude product was re-dissolved in pyridine, followed by the addition of acetic anhydride and 4-dimethylaminopyridine. After 24 h, the solvent was removed and the crude product was purified via silica column chromatography using ethyl acetate and hexane as the eluents. 1H NMR of AAM (CDCl3, 500 MHz): δ (ppm) 6.66&6.60 (d, J= 9.0 Hz, 1H, C(O)NHCH), 6.04&6.04 (d, 1H, J=1.9 Hz, NHCHCHO), 5.32-5.35&5.04-5.07 (dd, J=10.2, 4.2 Hz, 1H, CH2CHCHCH), 5.22&5.16 (t, J=9.9 Hz, 1H, CH2CHCHCH), 4.60-4.63&4.71-4.74 (m, 1H, NHCHCHO), 4.10-4.27 (m, 2H, CH2CHCHCH), 4.07 (m, 2H, C(O)CH2N3), 3.80-4.04 (m, 1H, CH2CHCHCH), 2.00-2.18 (s, 12H, CH3C(O)). 13C NMR (CDCl3, 500 MHz): δ (ppm) 170.7, 170.4, 170.3, 169.8, 168.6, 168.3, 167.5, 166.9, 91.5, 90.5, 73.6, 71.7, 70.5, 69.1, 65.3, 65.1, 62.0, 61.9, 52.8, 52.6, 49.9, 49.5, 21.1, 21.0, 21.0, 20.9, 20.9, 20.9, 20.8. ESI MS (m/z): calculated for C16H22N4O10Na [M+Na]+ 453.1, found 453.1.

Mass spectrometry. Electrospray ionization mass spectrometry (ESI-MS) was performed to confirm the molecular mass of the synthesized chemical compounds. A total of one purified sample was analyzed (n=1), as the analysis was conducted solely for compound identity verification. For sample preparation, the compound was dissolved in methanol or water based on the solubility, and 10 μL of the solution was directly injected into the mass spectrometer. The mobile phase consisted of 50% methanol containing 0.1% formic acid. Mass spectra were acquired using a Waters Quattro Ultima (Waters Corporation) operated in electrospray ionization positive mode (ESI⁺) with direct infusion. Raw data were acquired and processed using MassLynx software (Waters Corporation, v4.1) using default processing parameters, including automatic centroid detection, Savitzky–Golay smoothing, and automated peak picking. The resulting spectra were manually inspected, and the dominant molecular ion peak was annotated. The observed m/z value was compared with the calculated molecular mass of the target compound to validate compound identity.

General procedure of flow cytometry. Cells were prepared according to individual experimental designs. Flow cytometry analyses were performed on an Attune NxT flow cytometer (Thermo Fisher Scientific) equipped with 405 nm, 488 nm, 561 nm, and 640 nm lasers. Instrument settings were optimized using unstained and single-stained controls and kept constant within each experiment. Spectral compensation was calculated using single-color controls and applied uniformly to all samples. Acquisition thresholds were set on forward scatter (FSC) to exclude debris. Debris and dead cells were excluded based on FSC/SSC parameters and viability dye staining, and doublets were removed using FSC-A versus FSC-H gating. Cy5 fluorescence was detected in the APC channel (640 nm excitation, 660/20 nm emission filter). A minimum of 10,000 live singlet events were collected per sample. Data were analyzed using FlowJo (BD Biosciences). Mean fluorescence intensity (MFI) or percentage of positive cells was quantified as indicated in the figure legends. All experiments were independently repeated at least three times with similar results.

In vitro metabolic labeling of MEL cells. MEL cells were cultured in DMEM containing non-essential amino acids, 10% FBS, and 5% BSA at the density of 5×105/mL for 12 h. AAM or AAGal or AAGlu with a final concentration of 50 or 100 µM was added to the culture medium. At selected time points, cells were collected, washed with opti-MEM twice, and incubated with DBCO-Cy5 (10 µg/mL) and SYTO™ 85 Orange Fluorescent Nucleic Acid Stain (5 µM) at room temperature for 30 min. After washing, cells were analyzed on a flow cytometer. To initiate the differentiation of MEL cells, 2% DMSO and 50 µM iron citrate were added. The counts and size of cells were analyzed via a cell-counter, and hemoglobin was detected by analyzing UV absorption at 438 nm.

In vitro metabolic labeling of primary RBCs. Blood was collected from C57BL/6 or Balb/c mice, and RBCs were harvested after the removal of lymphocytes. To maintain the viability of mouse RBCs in vitro, DMEM media containing non-essential amino acids, 10% FBS, 5% BSA, and 10 g/L glucose were used. RBCs were seeded at 108/mL in 24-well plates in the presence of 50 or 100 µM AAM or AAGal. After 24 h, 107 RBCs were taken out, washed with Alsever’s solution twice, and incubated with DBCO-Cy5 (10 µg/ml) and SYTO™ 85 Orange Fluorescent Nucleic Acid Stain (5 µM) at room temperature for 1 h. Cells were washed and analyzed on a flow cytometer.

In vivo metabolic labeling of RBCs. C57BL/6 or Balb/c mice were intravenously injected with AAM (100 mg/kg) or PBS twice daily for 3 consecutive days. At 1, 3, 5, 7, 14, 21, 28, 35, 42, and 49 days post the last injection of AAM or PBS, blood was collected and stored in Alsever’s solution. After washing, cells were stained with DBCO-Cy5 (5 µg/mL) and SYTO™ 85 Orange Fluorescent Nucleic Acid Stain (5 µM) at room temperature for 1 h. After washing with Alsever’s solution for three times, samples were run on a flow cytometer. For some experiments, AAM (200 mg/kg) was i.p. injected for 2, 4, and 6 times with an interval of 12 h. For the analysis of RBC precursor cells in the bone marrow, at 48 h post the last injection of AAM, bone marrow cells in the tibia and femur of mice were collected and stained with DBCO-Cy5, anti-Ter119, anti-CD44, and fixable viability dye, prior to flow cytometry analysis. To detect azido-labeled cells in healthy tissues, tissues were sectioned with a thickness of 20 µm, stained with DBCO-Cy5 and DAPI for 20 min, washed with PBS for multiple times, and mounted onto a microscope slide with the addition of Prolong Gold, prior to confocal imaging.

Enrichment of azido-labeled RBCs. C57BL/6 mice were intravenously injected with AAM (100 mg/kg) or PBS twice daily for 3 consecutive days. At 7 days post the injection of AAM, RBCs were harvested, incubated with DBCO-desthiobiotin for 1 h, and then incubated with streptavidin-functionalized magnetic beads. Cells were then passed through a MACS cell separation column to remove unbounded RBCs (flow-through fraction). The magnetic bead-bounded RBCs were then eluted out using a solution of biotin.

Fluorescence imaging of RBCs. Blood was drawn from C57BL/6 mice at selected times post the injection of AAM or PBS, and kept in the Alsever’s solution. After washing, RBCs were incubated with 5 μg/mL DBCO-Cy5 at room temperature for 1 h, washed with Alsever’s solution for 5 times, and added to a glass coverslip. Samples were stored at 4℃ prior to fluorescence imaging.

RBC agglutination analysis. C57BL/6 mice were intravenously injected with AAM (100 mg/kg) or PBS twice daily for 3 days. At different times after the final AAM injection, blood was extracted from the tail of mice and diluted with the Alsever’s solution. A fraction of cells was directly imaged under a fluorescence microscope for morphology and agglutination analysis. Cells were also analyzed on a flow cytometer. The singlets were quantified from the FSC-A/FSC-H plot.

ATP level measurement. C57BL/6 mice were intravenously injected with AAM (100 mg/kg) or PBS twice a day for 3 days. At different times after the final AAM injection, blood was extracted from the tail of mice and washed with Alsever’s solution twice. The cell pellet was suspended in ATP-free water, followed by three freeze-thaw cycles to lyse RBCs. The ATP level in RBC lysates was measured using the ENLITEN® ATP Assay System Bioluminescence Detection Kit.

Resorufin assay of RBCs. C57BL/6 mice were intravenously injected with AAM (100 mg/kg) or PBS twice a day for 3 days. At different times after the final AAM injection, blood was extracted from the tail of mice and washed with Alsever’s solution twice. The cell pellet was resuspended in Alsever’s solution and incubated with resazurin (50 μg/mL) at 37℃ for 2 h, respectively. The resorufin product was detected on a plate reader (λex/λem = 560/590 nm). RBCs incubated with H2O2 at room temperature for 10 min were used as the negative control.

Osmotic fragility and mechanical fragility test. C57BL/6 mice were intravenously injected with AAM (100 mg kg⁻¹) or PBS twice daily for 3 consecutive days. Blood was collected 3 days after the final injection. For the AAM-treated group, red blood cells (RBCs) were separated into azido-positive and azido-negative populations using the above-described enrichment method for azido-labeled RBCs. These two fractions, together with RBCs from PBS-treated mice, were subjected to osmotic and mechanical fragility assays. For the osmotic fragility assay, 1×10⁶ RBCs were pelleted by centrifugation and resuspended in 100 μL NaCl solutions of varying concentrations. After incubation at room temperature for 30 min, samples were centrifuged to pellet intact RBCs. Hemolysis was quantified by measuring the absorbance of the supernatant at 415 nm and normalized to complete lysates achieved by incubation of RBCs in ACK lysis buffer. For the mechanical fragility assay, 1×10⁷ RBCs in 1 mL PBS from each group were transferred to tubes containing ten 4-mm glass beads and placed horizontally on an orbital shaker at 60 rpm. At the indicated time points, 100 μL aliquots were collected and centrifuged to remove intact RBCs. Hemolysis was determined by measuring the absorbance at 415 nm and expressed as a percentage of total lysis relative to ACK-treated samples.

In vivo targeting of RBCs. C57BL/6 mice were intravenously injected with AAM (100 mg/kg) or PBS twice a day for 3 days. At 3 days post the last injection of AAM, DBCO-Cy5 (5 mg/kg) was intravenously injected. At selected times, blood was collected and placed in the Alsever’s solution. After washing, cells were incubated with SYTO™ 85 Orange Fluorescent Nucleic Acid Stain (5 μM) at room temperature for 30 min, washed, and analyzed on a flow cytometer.

Western blot analysis. C57BL/6 mice were intravenously injected with AAM (100 mg/kg) or PBS twice daily for 3 days. Blood was collected at 7 days post the final injection of AAM or PBS. White blood cells were removed with Histopaque®-1077 according to the manufacturer’s protocol. RBCs were lysed with 0.1x PBS containing Halt™ Protease Inhibitor Cocktail at 4℃ for 30 min, followed by centrifugation at 21,000 g for 30 min and washing with 0.1x PBS containing Halt™ Protease Inhibitor Cocktail for 5 times to yield the RBC membranes. RBC membranes were lysed and incubated with alkyne-PEG4-biotin for overnight at 4℃. After washing with 0.1x PBS, proteins were loaded into SDS-PAGE gels for electrophoresis and subsequent transfer to the nitrocellulose membrane. The nitrocellulose membrane was blocked with the blocking buffer (5% non-fat milk in PBST) for overnight at 4℃, incubated with streptavidin-HRP for 30 min, washed with PBST for 5 times, and treated with HRP substrate for chemiluminescence imaging on an ImageQuant 800. Protein bands were also stained with Ponceau S. For some analyses, RBCs or proteins of RBCs were already conjugated with DBCO-desthiobiotin. Gel electrophoresis followed by desthiobiotin detection will be directly performed for those samples.

RBC lipid extraction and analysis. C57BL/6 mice were intravenously injected with AAM (100 mg/kg) or PBS twice daily for 3 days. Blood was collected at 7 days post the final injection of AAM or PBS. After removing the white blood cells with Histopaque®-1077, RBCs were incubated with DBCO-Cy5 (10 μg/mL) in Alsever’s solution for 1 h at room temperature. Cells were then washed with Alsever’s solution for three times and resuspended in a mixture of methanol and dichloromethane (3/7, v/v). The samples were then sonicated for 10 min. The protein precipitate was removed by centrifugation at 21,000 g for 10 min at 4℃. The lipid-containing supernatant was gently collected and analyzed on an HPLC.

Ex vivo retention of Cy5 on RBC surface. RBCs were isolated from C57BL/6 mice at 7 days post injections of AAM or PBS, and incubated with DBCO-Cy5 (5 μg/mL) for 1 h at room temperature. Cells were washed three times and resuspended in Alsever’s solution. An aliquot of sample was directly analyzed on the flow cytometer, and the remaining sample was stored at 4℃. At selected times, an aliquot of each sample was run on the flow cytometer.

In vivo fluorescence imaging of blood vessels and tumors. For B16F10 tumor study, C57BL/6 mice were intravenously injected with AAM (100 mg/kg) or PBS twice a day for 3 days. DBCO-Cy5 (5 mg/kg) was intravenously injected at 4 days post the final injection of AAM or PBS. Blood was drawn from the tail at selected times for fluorescence imaging. At 17 days post DBCO-Cy5 injection, 5x106 B16F10 cells were subcutaneously injected into the flank of C57BL/6 mice. Tumors were collected 7 days after inoculation and subjected to IVIS imaging. For 4T1 tumor study, Balb/c mice were intravenously injected with AAM (100 mg/kg) or PBS twice daily for 3 days. At 4 days post the final injection of AAM or PBS, 2×106 4T1 cells were subcutaneously injected into the flank of mice. After 3 days, DBCO-Cy5 (5 mg/kg) was i.p. injected. Blood was drawn at selected times for fluorescence imaging and flow cytometry analysis. IVIS imaging of mice was also performed at selected times. At 21 days post tumor cell inoculation, tumors and organs were harvested for IVIS imaging. During the 4T1 tumor imaging study, at selected times, blood was also collected for the analysis of Cy5 signal in the plasma. The blood cells were removed by centrifugation at 2,000 g for 10 min, and the supernatant was harvested for Cy5 fluorescence measurement on a plate reader.

Synthesis of DBCO-DOTA-Gd. 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA, 1.1 e.q.) and DBCO-PEG4-amine (1.0 e.q.) were dissolved in dimethyl sulfoxide, followed by the addition of N,N-diisopropylethylamine (DIPEA) (4.4 e.q.) and hexafluorophosphate azabenzotriazole tetramethyl uronium (1.0 e.q.). After 2 h, the solvent was removed and the residue was purified by preparative HPLC to yield DBCO-DOTA. DBCO-DOTA (1.0 e.q) and DIPEA (3.5 e.q.) were then dissolved in water, followed by the addition of Gd(NO3)3·6H2O (1.5 e.q.). After 4 h, the solvent was removed and the residue was purified by preparative HPLC to yield DBCO-DOTA-Gd.

MRI of mice. C57BL/6 mice were intravenously injected with AAM (100 mg/kg) or PBS twice a day for 3 days. At 4 days post the final injection of AAM or PBS, DBCO-DOTA-Gd (250 mg/kg) was intravenously injected. Mice were imaged before the injection and at different times after in a 9.4 T Bruker system (Bruker BioSpec 94/30 USR) using a 1H receive-only 2×2 mouse brain surface array coil. The mice were initially anesthetized with 2.5% isoflurane and maintained at 0.5-1.5% isoflurane throughout the experiment. Body temperature was held at 37°C by circulating warm water and monitored using a rectal thermosensor. The Small Animal Monitoring & Gating System (SA Instruments) was utilized for continuous monitoring of both respiration rate and body temperature, ensuring the respiration rate was controlled within the range of 35 to 65 per minute. For contrast-enhanced magnetic resonance angiography (CE-MRA) to compare blood vessel visualization, a 3D T1-weighted FLASH sequence was used with coronal slices to minimize tight of flight effects. Scan parameters were: TE = 2.11 ms, TR = 15 ms, readout bandwidth = 59 kHz, number of averages = 4, flip angle = 15, FOV = 23.25 × 14.23 × 13.27 mm3, matrix size = 200 × 180 × 72, and total scan time = 10 min. The same parameters were used for the scans.

DBCO-doxorubicin synthesis. DBCO-NHS and DIPEA were dissolved in acetonitrile, followed by the addition of hydrazine monohydrate. The mixture was stirred for 30 min. After removing the solvent, the crude product was purified via preparative HPLC to yield DBCO-hydrazide. DBCO-hydrazide and doxorubicin were then dissolved in anhydrous methanol, followed by the addition of trifluoroacetic acid. After the complete consumption of DBCO-hydrazide, the solvent was removed under reduced pressure. The crude product was purified via preparative HPLC to yield DBCO-doxorubicin.

DBCO-doxorubicin degradation. DBCO-doxorubicin was dissolved in buffers with a pH of 7.4, 6.5, and 5.5, respectively and incubated at 37oC. At selected times, 10 µL aliquots were run on an HPLC to determine the degradation kinetics of DBCO-doxorubicin.

In vivo targeting of DBCO-doxorubicin to RBCs. C57BL/6 mice were intravenously injected with AAM (100 mg/kg) or PBS twice a day for 3 days. At 3 days post the final injection of AAM or PBS, DBCO-doxorubicin (5 mg/kg) in a mixture of DMSO/PBS (1/9, v/v) was i.v injected. At 2 or 24 h post the injection of DBCO-doxorubicin, blood was collected from mice and stored in Alsever’s solution. After staining with SYTO™ 85 Orange Fluorescent Nucleic Acid Stain, cells were analyzed on a flow cytometer. Doxorubicin fluorescence signal in RBCs was detected via flow cytometry using the PE channel (488 nm excitation and 575 nm emission).

DBCO-PE synthesis and conjugation to RBC. PE (1.0 e.q.) was dissolved in PBS, followed by the addition of DBCO-PEG4-NHS (10 e.q.). The mixture was stirred at 4oC for overnight. After removing small molecules using the Sephadex G-25 resin column, the purified DBCO-PE was concentrated via ultracentrifugation with a 100 kDa ultra centrifugal filter. RBCs isolated from AAM- or PBS-treated mice were incubated with DBCO-PE in Alsever’s solution for 1 h at room temperature. After washing with Alsever’s solution for three times, cells were stained with a DNA dye and analyzed on a flow cytometer.

DBCO-OVA and DBCO-DNP-KLH synthesis and vaccination study. Ovalbumin (OVA) or dinitrophenyl–keyhole limpet hemocyanin (DNP-KLH) (1.0 equiv.) was dissolved in PBS, followed by the addition of DBCO-PEG4-NHS ester (10 equiv.). The reaction mixture was gently stirred at 4°C overnight. Unreacted small molecules were removed using a Sephadex G-25 desalting column. Purified DBCO-OVA was concentrated by ultrafiltration using a 30 kDa molecular weight cutoff centrifugal filter, while DBCO-DNP-KLH was concentrated using a 100 kDa molecular weight cutoff centrifugal filter. For the in vivo humoral response study, C57BL/6 mice were intravenously injected with AAM (100 mg/kg) or PBS twice daily for 3 consecutive days. Four days after the final AAM or PBS administration, mice were intravenously injected with DBCO-OVA or DBCO-DNP-KLH. Blood samples were collected at the indicated time points, and plasma was isolated by centrifugation at 2,000 g for 10 min to remove blood cells and platelets. Plasma samples were stored at -80 °C until analysis. After the final time point, all plasma samples were subjected to antibody titer analysis.

ELISA for antigen-specific IgG1 and IgG2a antibody titers. Unless otherwise specified, all procedures were performed at room temperature. OVA or DBCO- DNP-KLH was diluted in PBS to a concentration of 10 µg/mL and used to coat high-binding 96-well ELISA plates overnight at 4°C. Plates were washed three times with PBS and blocked with 2% (w/v) BSA in PBST (0.1% Tween-20 in PBS) for 1 h. After one wash with PBST, serially diluted plasma samples were added to the plates and incubated overnight at 4°C. Plates were then washed three times with PBST and incubated with biotin-conjugated anti-mouse IgG1 or IgG2a antibodies diluted in blocking buffer for 2 h. Following three additional washes with PBST, streptavidin-HRP diluted in blocking buffer was added and incubated for 30 min. After washing the plates three times with PBST, the HRP substrate was added and allowed to develop for 15 min before the reaction was terminated with 2 M sulfuric acid. Absorbance was measured at 450 nm. Antibody titers were defined as the lowest plasma dilution yielding a signal above baseline, or alternatively, OD values 450 were directly used for quantitative comparison.

Synthesis of DBCO-insulin. DBCO-ester-NHS was first synthesized. DBCO-PEG3-OH (1.0 e.q.) and succinic anhydride (2.0 e.q.) were dissolved in anhydrous pyridine, followed by the addition of 4-dimethylaminopyridine (1.0 e.q.). The mixture was stirred for overnight. After removing the solvent, the crude product was purified using preparative HPLC to yield DBCO-ester-COOH. DBCO-ester-COOH (1.0 e.q.) was then dissolved in dichloromethane, followed by the addition of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (1.2 e.q.) and N-hydroxysuccinimide (2.0 e.q.). The mixture was stirred overnight. After removing the solvent, the crude product was purified via silica gel column chromatography to yield DBCO-ester-NHS. The as-synthesized DBCO-ester-NHS (2.0 e.q.) and insulin (1.0 e.q.) were dissolved in PBS and stirred for overnight at 4oC. The crude product was purified via ultracentrifugation using a 3 k Da ultracentrifugal filter.

In vitro conjugation of DBCO-insulin to cells. 5×104 4T1 cells in 96-well plates were incubated with AAM (50 µM) or PBS for 24 h. Cells were detached with trypsin and incubated with DBCO-insulin (0.1 mg/mL) in FACS buffer (2% BSA in PBS) for 1 h at 4℃. After washing, cells were stained with rabbit anti-insulin primary antibody for 30 min at 4℃, and then incubated with Cy5-conjugated goat anti-rabbit secondary antibody for 30 min, prior to flow cytometry analysis. Similarly, RBCs isolated from AAM- or PBS-treated mice were incubated with DBCO-insulin in Alsever’s solution (0.1 mg/mL) for 1 h, followed by the staining with rabbit anti-insulin primary antibody and Cy5-conjugated goat anti-rabbit secondary antibody. Cells were also stained with a DNA dye. After washing, RBCs were analyzed on a flow cytometer.

STZ induced type 1 diabetes (T1D). To induce T1D, female C57BL/6 mice were injected with STZ (150 mg/kg). After monitoring the blood glucose levels for 7 days and confirming the onset of T1D (blood glucose level >220 mg/dL without fasting), mice were randomly divided into 4 groups. Mice were intravenously injected with AAM (100 mg/kg) or PBS twice daily for 3 days. Four days later, DBCO-insulin or insulin (10 IU/kg) was i.p injected. For the measurement of plasma insulin levels, blood was collected at 8 h post injection of DBCO-insulin or insulin and placed in heparin-coated tubes. After removing blood cells via centrifugation, the supernatant was harvested for the quantification of insulin using the insulin ELISA kit (Catalog 80-INSHU-E01.1).

Glucose tolerance test (GTT). Female C57BL/6 mice were fasted for 12 h, and DBCO-insulin or insulin (10 IU/kg) was i.p injected (counted as time 0). At selected times, blood was drawn from the tail, and the glucose level in the blood was measured using the Clarity BG1000 Blood Glucose Meter and Strips. At 3 h and 6 h, 1.5 g/kg glucose was i.p injected to each mouse. The blood glucose level was continuously monitored for 9 h with an interval of 0.5 h. After the GTT experiment, DBCO-insulin or insulin was injected every week for 2 more injections (3 injections in total). The body weight of mice was closely monitored.

Statistics and Reproducibility. Flow cytometry data were analyzed using FlowJo (BD Biosciences, v10.8.1). Statistical analyses and data visualization were performed using GraphPad Prism (GraphPad Software, v9.5.1). For in vitro studies, each experiment included at least three biological replicates, and technical replicates were included where appropriate. For in vivo experiments, the number of animals (n) per group is specified in the corresponding figure legends. Animals were randomly assigned to experimental groups when applicable. Representative images (e.g., confocal microscopy, fluorescence imaging, histology) are shown from one experiment, and similar results were observed in at least three independent experiments or independent biological replicates. Statistical analyses were performed as described in the corresponding figure legends. Data are presented as mean ± SD. Exact p values and statistical tests are indicated in the figures or figure legends.

Data Availability. All data provided in this study can be found in the main text, figures, supplementary information, and source data files.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Hua Wang and Co-workers in this paper have investigate the development and possible use of RBC for coupling different kind of agents of possibly diagnostic or therapeutic interest. The idea is not original since many labs have already produced and used RBC coupled to agents of interest. A number of reviews are available. Hua Wang et All. have investigate a possible glycan labelling producing data obtained in vitro and in vivo on murine RBC. Metabolic glycan labeling has been widely explored for the engineering of cancer cells, immune cells, stem cells, and other types of cells as highlighted in the reference list correctly reported in the manuscript. This approach is of potential general interest and a number of applications are shown.\nThe major limitation of the manuscript is the use only of mice RBC without any evidence that similar results could be translated to human RBC. This is not a trivial difference that should at least be discussed.\nThe second major concern involve the possible induced immunogenicity of the generated constructs. During the years it has been shown that aptens or peptides/proteins coupled to the RBC membrane can become immunogenic after one or more administrations in vivo. In some cases the modality of coupling and the nature of the agent coupled can instead induce immunotolerance.\nTo overcome these limitations potentially therapeutic agents have been coupled to RBC membranes by a number of different approaches (i.e. https://doi.org/10.3390/pharmaceutics12050440) but also encapsulated into the RBC to prevent drug inactivation by eventually generate antibodies (https://doi.org/10.1080/17425247.2020.1822320). These modalities should be mentioned since relevant to the scope of the paper.\nA further question is related to the possibility of activate the RBC membrane in vivo. Apparently this is a new approach but others have previously administered in vivo agents that modify the RBC membrane with success (Blood, Vol 93, No 1 (January 1), 1999: pp 376-384).\nThe Discussion section should consider not only the potential benefits but also the limits of the approach (some examples are briefly mentioned above) and spend some time to provide to the reader a more complete view of the approaches developed by others in the field of RBC use as carriers for therapeutic or diagnosic agents.\nIt is not fear to write “Existing RBC engineering approaches are limited to isolated RBCs under ex vivo conditions, and the whole process of isolating RBCs, modifying RBCs ex vivo, and infusing engineered RBCs is time-consuming and costly, often causes damage to RBCs, and suffers from a high risk of infections.(Ref. 9,17-19)”. A recent paper published il Lancet Neurology (DOI: 10.1016/S1474-4422(24)00220-5) reports data obtained in human patients with a rare disease highlighting safety and efficacy of a RBC-based drug delivery system in a large phase 3 clinical trial. Furthermore, references 17-19 are not appropriate referring to transfusion products not to engineered RBC.\nMinor points:\nExtended Fig. 3a and 3b: please explain the meaning of Cy5Fluorescence intensity axes in Fig.3a per µL blood. Is this figure showing the n. of Cy5+ cells or fluorescence intensity?\nWith respect to the percentage of RBC-AAM (and WBC-AAM) can you explain figures close to about 100% (Ext Fig 3b) when the main text explain that in vivo at 24h from AAM administration labelled RBC are 4.2%? If the Fig.3b represent the total number of RBC (and WBC) it is not clear at 7.5 days if in circulation we still have all labelled RBC or if 100% RBC is represented by new RBC entering in circulation to replace RBC-AAM that are removed from circulation as shown in Ext. Fig. 3a at day 7.5. Please comment.\nExtended data Fig.4: please explain Fig.4b. % refer to labelled cells or total cells? If the figure refer to total cells this is not representative of the successful labelling of RBC precursors but simply of RBC precursors distribution number upon AAM labelling versus PBS. Please clarify\nIntraperitoneal injections: the dose administered to increase the % of labelled RBC to max 14% is really very high totaling about 1.2g/Kg of AAM administered! Please comment.\nIn vivo toxicity: The determination of RBC main properties obtained from cells of animals receiving iv AAM are of limited interest since only a small percentage of said RBC are modified by AAM under the experimental conditions used. In other words measurements of ATP or NAD/NADH are representative of all RBC present in the samples and not of the labelled RBC. This should be commented. In addition, Fig. 4j clearly shows that heart and kidney are significantly labelled by AAM.\nIn vivo targeting of DBCO cargo: please comment Extended Data Figure 6. The differences among day 0 and day 1; in addition explain while day 5 Cy5 Fl ratio appears to decrease respect day 3.\nLong-term fluorescence imaging: labelled RBC have been administered i.p. and not i.v., please comment. Is this modality more appropriate for targeting/labelling implanted tumor cells?\nMRI imaging of brain blood vessels: Assuming that Gd coupled to RBC is stable were Gd ends when disappear from circulation? Is this removed in spleen and liver? Does cause any kidney toxicity? Please address these comments.\nIn vivo conjugation of drugs to RBCs: DBCO-doxorubicin intravenously administered can label only about 1% of RBC at 2 hours and is not significant versus the administration of PBS. These results are of very limited interest and doesn’t prove any potential therapeutic benefit. The same apply to coupling of PE. In this case DBCO-functionalized PE is added ex-vivo for two hours to AAM activated RBC. The same apply to DBCO-insulin detected with rabbit anti insulin and Cy5-conjugated goat anti-rabbit secondary antibody on RBC after an ex-vivo incubation of two hours. The difference versus mice receiving PBS is not significantly different. In vivo experiments with peritoneal administration of DBCO-insulin in AAM treated STR mice show a limited effect only at one time point questioning the benefit of this claimed therapeutic treatment. This should be commented in more details highlighting the limitations of the approach.", "reviewer_2": "The study by Yusheng Liu et al describe a novel approach for RBC’s engineering with the potential application in imaging and drug delivery.\nOverall, the stud is well designed and executed, following are few comments that could further benefit the work:\n1. RBCs are critical for gas exchange, endothelial functions, platelet reactivity, thrombosis and haemorrhage, binding to pathogens, scavenging chemokines. Furthermore, the elasticity of RBC’s is essential for vascular functions specially at the presence of prooxidants s. These functions require through testing to ensure the competence of RBCs after azido tagging and cargo incorporation. Tests such as membrane integrity tests (osmotic and Mechanical), oxidative haemolysis assay, Ektacytometry, and RBC’s indices such as MCV, RDW, MCH and MCHC; can augment the safety profile of the proposed strategy.\n2. The main proposed usages mentioned in the studies are, imaging and drug delivery. For imaging, the reasoning for having a long-term tagging needs to be justified compared to classical imaging techniques which relies on short acting contrast agents. Similarly for RBCs as a drug carrier, more discussion is needed to justify the advantages of this pathway compared to using exogenous carriers (e.g. nanoparticles).\n3. The study demonstrated improved pharmacokinetics for drugs like insulin, however, the release kinetics of conjugated drugs from RBCs are not thoroughly characterized. This is critical for ensuring controlled and predictable therapeutic outcomes.", "reviewer_3": "This manuscript from the Wang lab describes the labeling of red blood cells (RBCs) with azido sugars for various potential downstream applications. The authors convincingly show that RBCs can be metabolically labelled with ManNAz (termed AAM here) both in vitro and in vivo. Additionally, the subsequent bioorthogonal reaction (SPAAC) can be performed in vivo as well. All of this confirms data from the Bertozzi lab and others concerning these sugars and SPAAC reaction. While the levels of the labeling are modest, the interesting and new feature here is that the azide-sugar persists on the cell surface of RBCs much longer than on other cells and tissues, allowing for a level of selectivity based on the timing between AAM and DBCO delivery. Using this feature the authors go on to show that they can detect differences in vascularization by fluorescence and image blood vessels by MRI. Finally, they conjugate pharmaceutical cargos to the RBCs and nicely show that insulin can act longer when appended to RBCs. Again, the magnitude of the differences here are mostly small, but I believe this is a potentially interesting application of metabolic engineering that is worth further exploration, particularly in terms of cargo circulation and delivery. I am supportive of publication. I do have two comments that should be addressed first.\nComment #1 - I suggest that the authors are more explicit about achieving RBC selectivity by waiting longer between AAM delivery and subsequent treatment with DBCO. I understand that they talk about the unique retention of azides on RBCs versus other tissues several times in the paper. However, I think this could be made more clear for non-experts, so that readers do not come away with the impression that other tissues are not labeled at all.\nComment #2 - How did the authors detect DOX on cells in Figure 9d? Was this antibody-based as well. I did not see the details for this experiment.", "reviewer_4": "The submitted study reports in vivo metabolic glycan labeling of red blood cells (RBCs) using systemically administered azido-sugars. The authors claim that RBCs are tagged with azides as cellular surface glycans (glycoproteins and glycolipids). The authors showed the surface azido tags on RBCs persist >42 days, whereas tags on leukocytes and other cells rapidly decay. The labeled RBCs can capture DBCO-linked cargos by click chemistry, prolonging a cargo’s circulation from hours to >35 days. As examples, one dose of azido-sugar plus a DBCO-gadolinium agent yields months-long brain-vasculature MRI, and linking insulin to tagged RBCs extends its blood half-life. In summary, the paper claims a stable in vivo RBC-labeling platform that dramatically extends cargo persistence and enables new imaging and drug-delivery applications.\nWhile the study demonstrates solid technical execution and offers long-lived labeling of circulating RBCs using azido sugars and click chemistry, it fundamentally builds upon well established strategies already reported by the same research group. The core methodology, systemic delivery of peracetylated azido-monosaccharides for metabolic labeling and subsequent conjugation of DBCO-bearing molecules via SPAAC, has been applied previously to dendritic cells, platelets, and adipocytes. In each case, similar labeling and in vivo targeting were made. The differencein this RBC study from previous is not the chemical mechanism, but merely the target cell type (RBCs) and the extended persistence of the azido label. While the longer half-life of labeled RBCs is notable (∼42 days vs ∼4 days for platelets), this reflects more a known biological property of RBCs than an engineered advance. Thus, the central novelty lies in demonstrating expected behavior in a new context. Moreover, the applications presented, such as prolonged imaging or drug delivery, are anticipated outcomes of this platform, not surprising or mechanistically new.\nAlthough the study convincingly demonstrates that azido groups persist on the surface of circulating RBCs for over 42 days, it does not adequately establish that this labeling strategy is selective enough for practical applications in targeted imaging or drug delivery. The authors argue that azide tags decay rapidly in leukocytes and other nucleated blood cells within 3 days, thereby enabling selective RBC targeting using DBCO-functionalized molecules if administered after this window. However, this argument overlooks a more critical issue: whether normal tissues and vasculature are also being labeled and whether that labeling persists to a degree that compromises selectivity. In particular, Figure 5i-j shows detectable fluorescence signals in normal tissues (liver, spleen, etc.) after DBCO-Cy5 injection, even in the AAM-treated group. Although the signal is lower than in RBCs, it is not negligible, and there is no quantification relative to background or functional implications of this residual labeling. The authors use a “wait 3 days post AAM” strategy to reduce leukocyte labeling, but it remains unclear whether this delay also fully eliminates labeling in non-blood tissues. The Western blot and confocal data in Fig. 4i-j are helpful but limited, they do not resolve whether low-level but functionally significant tagging persists. Furthermore, the authors' own prior studies on metabolic labeling of platelets, adipocytes, and dendritic cells reported relatively broad azido labeling, including some tissue types. The current claim that RBCs uniquely retain azido tags while all other tissues rapidly clear them seems inconsistent with those earlier findings, suggesting either differing experimental standards or unexplored biological variability. This inconsistency undermines the claim of RBC-specific labeling and suggests that more rigorous, quantitative tissue-distribution studies are needed.\nOverall, The study is technically well-conducted and shows promise for translational applications, but it does not introduce a sufficiently novel concept or mechanism to meet the publication standards of Nature Communications. The core advances are too directly on a sequence of prior works from the same group using near-identical strategies. There is no demonstration of fundamentally new biological insight, molecular mechanism, or chemical methodology that would justify publication in a journal prioritizing broad conceptual advances. In addition, the study does not sufficiently demonstrate RBC-specific labeling or targeting, and several results raise the possibility of non-negligible off-target effects in normal tissues. This undermines the claimed value of the system for selective imaging or drug delivery. Additionally, the conclusions appear partially inconsistent with earlier work from the same group on metabolic labeling of other cell types. Without stronger and more systematic evidence of selectivity, this work does not meet the robustness or clarity needed for publication in Nature Communications."}, "author_response": "Point-by-point reply to Reviewers' comments\n(All responses were colored in blue; all changes in the manuscript were highlighted in\nyellow)\nReview #1\nHua Wang and Co-workers in this paper have investigated the development and possible use\nof RBC for coupling different kind of agents of possibly diagnostic or therapeutic interest. The\nidea is not original since many labs have already produced and used RBC coupled to agents of\ninterest. A number of reviews are available. Hua Wang et al. have investigated a possible\nglycan labelling producing data obtained in vitro and in vivo on murine RBC. Metabolic glycan\nlabeling has been widely explored for the engineering of cancer cells, immune cells, stem cells,\nand other types of cells as highlighted in the reference list correctly reported in the manuscript.\nThis approach is of potential general interest and a number of applications are shown.\nResponse: We thank the Reviewer for the positive comments, and addressed the remaining\ncomments in the revised manuscript.\n1. The major limitation of the manuscript is the use only of mice RBC without any evidence\nthat similar results could be translated to human RBC. This is not a trivial difference that should\nat least be discussed.\nResponse: We appreciate this comment from the Reviewer. We have attempted to test the\nmetabolic labeling of human RBCs, but have been experiencing trouble (mostly on IRB side)\nin obtaining fresh human RBCs. For now, we may not be able to fully address this comment\nexperimentally, although we are fairly confident that the metabolic glycan labeling should work\nfor human RBCs. We have now added some discussion on the applicability towards human\nRBCs in the revised manuscript: “While we have not tested metabolic glycan labeling of human\nRBCs, the well-known presence of glycoproteins and glycolipids in human RBCs adds to the\nfeasibility. Indeed, human RBCs mainly express Neu5Ac (AAM labeling pathway) while\nmurine RBCs express both Neu5Ac and Neu5Gc,51 suggesting that AAM-mediated metabolic\nglycan labeling could be more efficient in human RBCs. Given the much longer lifespan of\nhuman RBCs (~120 days), we also anticipate that azido-labeled RBCs could retain longer in\nthe bloodstream than azido-labeled murine RBCs. Future studies on in vitro and in vivo labeling\nof human RBCs are needed to fully assess the safety and translational potential of the RBC\nlabeling and targeting technology.” [Ref. 51: Varki, A. Loss of N-glycolylneuraminic acid in\nhumans: mechanisms, consequences, and implications for disease. Proc. Natl Acad. Sci. USA\n98, 5379–5386 (2001)]\n2. The second major concern involve the possible induced immunogenicity of the generated\nconstructs. During the years it has been shown that aptens or peptides/proteins coupled to the\nRBC membrane can become immunogenic after one or more administrations in vivo. In some\ncases the modality of coupling and the nature of the agent coupled can instead induce\nimmunotolerance.\nResponse: We thank the Reviewer for this comment. In the current manuscript, we mainly\nreported small-molecule DBCO-functionalized agents (fluorophores, gadolinium, insulin)\nwhich are generally lowly immunogenic. Indeed, we attempted to utilize the RBC labeling and\ntargeting technology for vaccine applications, with a hope that the longer retention of antigens\nin the bloodstream may prolong the antibody response. We tried to conjugate DBCO-DNP-\nKLH and DBCO-OVA, two representative antigens, to circulating RBCs and monitored the\nantibody titers in the blood over time. As shown in the new Fig. S9, none of the conjugation\ngroups showed enhanced antibody responses in comparison with the control groups.\nWe have now added Fig. S9 and the following text in the revised manuscript: “We are aware\nof the concern on the potential immunogenicity of cargos being conjugated to circulating RBCs.\nIn separate studies, we attempted to conjugate DBCO-functionalized 2,4-dinitrophenylated\nHemocyanin, Keyhole Limpet (DNP-KLH) and DBCO-ovalbumin (OVA), two representative\nantigens, to RBCs in vivo and monitor the antibody titers over time. As a result, the in vivo\nconjugation of DNP-KLH and OVA antigens failed to exert any enhancement on the antibody\nresponses compared to the control groups (Fig. S9), which lessens the concern of potent\nimmunogenicity of RBC-conjugated cargos in our metabolic labeling approach.”\n#C(\n#C!\n#CD\n#CA\n#C#\n! \" #! A# AB\n)GHI-KLIM-GNMOP4N-ON54SMOLN\nFigure S9. In vivo targeting of antigens to circulating RBCs does not enhance the antibody\nresponses. (a) Timeframe of DNP-KLH vaccination study. C57BL/6 mice were i.v. injected\nwith AAM or PBS twice daily for three consecutive days. After labeling, mice were i.v. injected\n<4MOM-H;L:OMN9-#8P7\n99=->-)?@AB)CaBbcd\na?e->-)?@AB)CaBbcd CD(\nCD#\nCDA\nCDC\n! \"# A\" AB\n)GHI-KLIM-GNMOP4N-ON54SMOLN\nGA<P;-:L9-C8#)7\nNLOM?>O=-CCC\"-MG\n@@A-B-)Ca7E)cdEefg dCK-B-dCK\ndCK-B-)Ca7E)cdEefg\nAD(\nADC\n\"D(\n\"DC\nCD(\nCDC\n! \"# A\" BC #A\n\"1M/+.-I+C(#G) 8-5P7S54+CCC\"+PO\nV:;<V:; @@A<G:=)>)?@\nV:;<G:=)>)?@\nCD\"C\nCDC)\nCDC(\nCDC#\nCDCA\nCDCC\n! \"# A\" BC #A\nGOBC+a-CP+G:=)>)?@+58EcdP5-8\n4APO1-M/.-CI#+G :/75V87S-CCC\"-54\na\nb c\nd\ne f\n;<=>;<= BBC>+<?G@GAB\n;<=>+<?G@GAB\n+4aE-c/E5-+<?G@GAB-7:def57/:\nwith DBCO-DNP-KLH, and serum antibody titers were monitored over time. (b) Serum anti-\nDNP-KLH IgG1 antibody titers at indicated time points. (c) Serum anti-DNP-KLH IgG2a\nlevels as determined by ELISA. (d) Timeframe of ovalbumin (OVA) vaccination study.\nC57BL/6 mice were i.v. injected with AAM or PBS twice daily for three consecutive days.\nAfter labeling, mice were i.v. injected with DBCO-OVA, and serum antibody titers were\nmonitored over time. Shown are anti-OVA (e) IgG1 and (f) IgG2a levels at indicated time\npoints. ELISA data are presented as OD values at the indicated serum dilution. All the\n450\nnumerical data are presented as mean ± SD.\n3. To overcome these limitations potentially therapeutic agents have been coupled to RBC\nmembranes by a number of different approaches\n(i.e. https://doi.org/10.3390/pharmaceutics12050440) but also encapsulated into the RBC to\nprevent drug inactivation by eventually generate antibodies\n(https://doi.org/10.1080/17425247.2020.1822320). These modalities should be mentioned\nsince relevant to the scope of the paper.\nResponse: We appreciate this comment from the Reviewer. We have now expanded the\ndiscussion on prior RBC engineering methods and cited the relevant references: “Non-covalent\nmethods involve physical adsorption of cargos onto RBC membranes via electrostatic\ninteractions11,22,23 or binding of a targeting ligand to RBC receptors (e.g., Ter119).13,24-26 The\nformer is limited by the weak interactions between cargos and RBCs and the need of custom-\ndesigned adsorbable structures,11,22,23 while the latter is limited by the lack of specificity and\nlow abundance of RBC receptors.13,24-26” “While direct encapsulation of cargos into RBCs\nhas been extensively explored, most approaches rely on ex vivo engineering of RBCs,48,49\nwhich inevitably inherits the common issues in RBC stability, infections, costs during the\nisolation, storage, ex vivo manufacturing, and infusion processes. Several prior studies utilized\ni.v. injection of N-hydroxysuccinimide (NHS)-functionalized agents for covalent coupling to\ncirculating RBCs,50 but the non-specific non-bioorthogonal NHS-amine chemistry presents a\ntremendous safety concern by non-specifically tagging all types of proteins, lipids, sugars, and\nnucleic acids”.\nA. J., Brenner, J. S. & Muzykantov, V. R. Vascular drug delivery using carrier red\nblood cells: focus on RBC surface loading and pharmacokinetics. Pharmaceutics 12,\n440 (2020).\nenzyme-loaded red blood cells. Expert Opin. Drug Deliv. 18, 43–54 (2021).\nEvaluation of biochemical changes during in vivo erythrocyte senescence in the dog.\nBlood 93, 376–384 (1999).\n4. A further question is related to the possibility of activate the RBC membrane in vivo.\nApparently this is a new approach but others have previously administered in vivo agents that\nmodify the RBC membrane with success (Blood, Vol 93, No 1 (January 1), 1999: pp 376-384).\nResponse: We thank the Reviewer for this comment. Previous work by Rettig et al. (Blood,\n1999) investigated the biochemical changes occurring during RBC senescence in dogs. They\nused NHS-biotin to modify circulating RBCs with biotin using the NHS-amine chemistry, with\na goal of retrieving the RBCs for ex vivo analysis. While their goal was to tag and isolate RBCs,\ntheir data indicated that the biotinylation of RBCs did not notably alter the cellular function.\nAs our bioorthogonal click chemistry approach is more selective than NHS-amine chemistry,\nwe anticipate that our approach will also be safe and do not alter RBC functions, as supported\nby our data in this manuscript.\nAlso, in response to another Reviewer’s comment regarding potential perturbation of\nmembrane integrity or function due to our labeling strategy, we have now included additional\nexperimental data demonstrating that the metabolic tagging of RBCs with azido groups does\nnot alter RBC membrane properties such as mechanical and osmotic fragility (Fig. S6).\n'!!\nC!\nB!\nA!\n#!\n!\n!\"! !\"# !\"A !\"B !\"C '\"!\n()*+,*-./-M)*N)2NP/(4N5S7\nFigure S6. In vivo metabolic glycan labeling of RBCs does not affect the osmotic and\nmechanical integrity of RBCs. C57BL/6 mice were i.v. injected with AAM or PBS twice\ndaily for three consecutive days. Four days after the final injection, peripheral blood was\ncollected and RBCs were isolated for functional assessment. (a) Osmotic fragility assay\nshowing percentage of hemolysis as a function of NaCl concentration. (b) Mechanical fragility\nassay measuring hemolysis following rotational stress over time. Data are presented as mean ±\nSD. Hemolysis was calculated as the ratio of supernatant absorbance at 415 nm (hemoglobin)\nto the absorbance at 415 nm obtained from an equal number of RBCs fully lysed with the ACK\nlysis buffer.\n'!!\nC!\nB!\nA!\n#!\n!\n!\"! !\"# !\"A !\"B !\"C '\"!\n()*+,*-./-M)*N)2NP/(4N5S7\n;M;:4)9,8N2)NSN\n<<=N5<>M?,@7\n\"!!\n<<=N5<>M?,A7 778.179,:M;S\nBCa '! 778.179,:M<S\n=>?\n&!\nA!\n#!\n!\n! \"# #A B& A' &!\n()*+*,)-.*,/M.12)P4RS\nR,RCB)/M2.A).@.\na b\n5. The Discussion section should consider not only the potential benefits but also the limits of\nthe approach (some examples are briefly mentioned above) and spend some time to provide to\nthe reader a more complete view of the approaches developed by others in the field of RBC\nuse as carriers for therapeutic or diagnostic agents.\nResponse: We thank the Reviewer for this comment. We have now revised the Discussion\naccordingly. Specifically, we have now included discussions on the applicability of our\nlabeling and targeting technology to human RBCs, the possibility of immune responses arising\nfrom repeated administrations of cargos, and limitations related to the diversity and size of\ncargos that can be efficiently conjugated and delivered using our platform: “While we have not\ntested metabolic glycan labeling of human RBCs, the well-known presence of glycoproteins\nand glycolipids in human RBCs adds to the feasibility. Indeed, human RBCs mainly express\nNeu5Ac (AAM labeling pathway) while murine RBCs express both Neu5Ac and Neu5Gc,51\nsuggesting that AAM-mediated metabolic glycan labeling could be more selective and efficient\nin human RBCs. Given the much longer lifespan of human RBCs (~120 days), we also\nanticipate that the azido-labeled RBCs could retain longer in the bloodstream than azido-\nlabeled murine RBCs. Future studies on in vitro and in vivo labeling of human RBCs are\nneeded to fully assess the safety and translational potential of the RBC labeling and targeting\ntechnology.” “Our in vivo RBC metabolic labeling approach offers a minimally invasive\nand modular strategy for RBC functionalization, but several limitations warrant attention.\nDifferences between murine and human RBCs may impact the overall labeling efficiency and\n;M;:4)9,8N2)NSN\n<<=N5<>M?,@7\n\"!!\n<<=N5<>M?,A7 778.179,:M;S\nBCa '! 778.179,:M<S\n=>?\n&!\nA!\n#!\n!\n! \"# #A B& A' &!\n()*+*,)-.*,/M.12)P4RS\nR,RCB)/M2.A).@.\na b\ncargo retention time, and the potential immunogenicity upon repeated dosing of cargos remains\nto be fully assessed. Also, the amounts of cargos, especially high-molecular-weight cargos, that\ncan be conjugated to circulating RBCs could still be limited due to the definite number of azido\ntags per RBC. Compared to existing RBC engineering methods, our approach bypasses the\nprocess of isolating RBCs, engineering RBCs ex vivo, and infusing the engineered RBCs, but\nfurther optimization of the labeling specificity and efficiency is needed to realize the\ntranslational potential.”\n6. It is not fear to write “Existing RBC engineering approaches are limited to isolated RBCs\nunder ex vivo conditions, and the whole process of isolating RBCs, modifying RBCs ex vivo,\nand infusing engineered RBCs is time-consuming and costly, often causes damage to RBCs,\nand suffers from a high risk of infections.(Ref. 9,17-19)”. A recent paper published il Lancet\nNeurology (DOI: 10.1016/S1474-4422(24)00220-5) reports data obtained in human patients\nwith a rare disease highlighting safety and efficacy of a RBC-based drug delivery system in a\nlarge phase 3 clinical trial. Furthermore, references 17-19 are not appropriate referring to\ntransfusion products not to engineered RBC.\nResponse: We appreciate this comment from the Reviewer. We have now updated the\nIntroduction: “Existing RBC engineering approaches mostly rely on isolating and modulating\nRBCs under ex vivo conditions, and the whole process of isolating RBCs, modifying RBCs ex\nvivo, and infusing engineered RBCs is time-consuming and costly, could cause damage to\nRBCs, and suffers from a risk of infections.9,17-19 Significant progress has indeed been made to\naddress these issues and bring RBC-based drug delivery systems to clinical trials.20,21 In\nparallel, we envision direct in vivo engineering of RBCs will provide new possibilities for\nunderstanding RBC biology and developing enhanced diagnostics and therapies for a variety\nof diseases” We have also updated the references accordingly.\nThe Lancet 370, 415-426 (2007)\nDrug Deliv. 11, 677–687 (2014).\nPhysiol. 10, 1070 (2019).\nphosphate in children with ataxia telangiectasia (ATTeST): a multicentre, randomised,\ndouble-blind, placebo-controlled phase 3 trial. Lancet Neurol. 23, 871–882 (2024).\nphosphate in children with ataxia telangiectasia (ATTeST): a multicentre, randomised,\ndouble-blind, placebo-controlled phase 3 trial. Lancet Neurol. 23, 871–882 (2024).\nMinor points:\n7. Fig. S3a and S3b: please explain the meaning of Cy5 Fluorescence intensity axes in Fig.S3a\nper µL blood. Is this figure showing the n. of Cy5+ cells or fluorescence intensity?\nResponse: Fig. S3a presents the total Cy5 fluorescence intensity per microliter of blood, as\ncalculated by ‘# of RBCs (or WBCs) per µL × mean Cy5 fluorescence intensity of RBCs (or\nWBCs)’. Fig. S3b shows the percentage of RBCs or WBCs among all the blood cells at\ndifferent time points. We have updated the figure captions to minimize confusion: “(a) Total\nCy5 fluorescence intensity of RBCs and WBCs that were harvested at different times and\nstained with DBCO-Cy5, as calculated by # of RBCs (or WBCs) per µL × mean Cy5\nfluorescence intensity of RBCs (or WBCs). (b) Percentages of RBCs and WBCs among blood\ncells at different times post injections of AAM or PBS.”\n8. With respect to the percentage of RBC-AAM (and WBC-AAM) can you explain figures\nclose to about 100% (Fig. S3b) when the main text explain that in vivo at 24h from AAM\nadministration labelled RBC are 4.2%? If the Fig. S3b represent the total number of RBC (and\nWBC) it is not clear at 7.5 days if in circulation we still have all labelled RBC or if 100% RBC\nis represented by new RBC entering in circulation to replace RBC-AAM that are removed from\ncirculation as shown in Fig. S3a at day 7.5. Please comment.\nResponse: Fig. S3b shows the percentage of RBCs or WBCs among all the blood cells. The\npurpose of presenting Fig. S3b is to show that AAM treatment did not alter the blood cell\nfractions (i.e., not toxic). Figures in the main text refer to the percentages of azido-labeled\nRBCs among all the RBCs, showing the metabolic labeling efficiency of RBCs.\n9. Fig. S4: please explain Fig. S4b. % refer to labelled cells or total cells? If the figure refer to\ntotal cells this is not representative of the successful labelling of RBC precursors but simply of\nRBC precursors distribution number upon AAM labelling versus PBS. Please clarify\nResponse: Similarly, Fig. S4b refers to total cells. The purpose is to show that AAM treatment\ndid not change the populations of RBC precursors (i.e., not toxic). To avoid confusion, we have\nupdated the Y axis of Fig. S4b.\n80\n60\n40\n20\n0\no o y o c C\nPr B a s P ol Ort hr R eti R B\nFig. S4b. Percentages of different RBC precursor cells among total erythroid cells at 2 days\npost injections of AAM or PBS.\n10. Intraperitoneal injections: the dose administered to increase the % of labelled RBC to max\n14% is really very high totaling about 1.2g/Kg of AAM administered! Please comment.\nResponse: For i.v. injections, 100 mg/kg per dose (6 doses in total) led to ~10% azido-labeled\nRBCs. For i.p., a higher dose could be used so we tested 200 mg/kg per dose. We believe the\nReviewer is concerned about the safety of a seemingly high dose of AAM. Indeed, a dosage of\n50-200 mg/kg AAM or similar azido-sugars with a total number of 3-9 doses has been\ncommonly used in previous studies by other groups and our own group.\n11. In vivo toxicity: The determination of RBC main properties obtained from cells of animals\nreceiving iv AAM are of limited interest since only a small percentage of said RBC are\n)%\n(\ntnecreP\nb\nAAM\nPBS P=0.0673\nP=0.1038\nP=0.3988\nP=0.7910 P=0.6281\nP=0.7651\nfo\negatnecreP\nsllec\ndiorhtyre\nlatot\nmodified by AAM under the experimental conditions used. In other words measurements of\nATP or NAD/NADH are representative of all RBC present in the samples and not of the\nlabelled RBC. This should be commented.\nResponse: We appreciate the Reviewer’s comment. We did receive a lot of questions on the\nsafety of our labeling technology, so the overall safety data will be helpful to our readers. Also,\nalthough only a fraction (~10%) of circulating RBCs is labeled under our current conditions,\nthis proportion is still substantial enough that bulk measurements (ATP, NAD/NADH) would\nlikely capture any major systemic or off-target effects of AAM treatment on overall RBC\nphysiology. We agree with the Reviewer that characterizing the metabolic state of only the\nlabeled RBCs would provide more specific insight. However, the process of purifying azido-\nlabeled RBCs itself would alter RBC metabolism and introduce artifacts. Instead, we have\nadditionally assessed RBC indices (MCV, MCH, MCHC, RDW), which revealed negligible\ndifferences between AAM and control groups. If AAM had notable effects on RBC physiology,\nchanges in these indices would be expected even with ~10% labeled cells.\nWe have added Fig. S7 and the following text in the revised manuscript: “RBC indices\nincluding RBC counts, Hemoglobin concentration, hematocrit percentages, mean cell volume\n(MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration\n(MCHC), and red cell distribution width (RDW) (Fig. S7a-g), as well as RBC morphology\n(Fig. S7h), showed negligible differences between AAM and PBS groups. These data\ndemonstrated that AAM-mediated metabolic labeling induced negligible changes in RBC\nmembrane fragility and RBC functions.”\n*+\n)\n(\n'\nC\n#B \"A !A\nFigure S7. In vivo metabolic glycan labeling of RBCs does not alter hematological indices\nor morphology of RBCs. C57BL/6 mice were i.v. injected with AAM or PBS twice daily for\nthree consecutive days. Four days after the final injection, peripheral blood was collected for\nhematological analysis. Quantified RBC parameters include (a) RBC count, (b) hemoglobin\nconcentration, (c) hematocrit, (d) mean corpuscular volume (MCV), (e) mean corpuscular\nhemoglobin (MCH), (f) mean corpuscular hemoglobin concentration (MCHC), and (g) red cell\ndistribution width (RDW). (h) Representative RBC morphology assessment for individual\nP21M(C*L.-,\",\n)H\n)*\n(\n'\n&\n#B \"A !A\n:987MS54P2L1ML.-,\nH&\n*&\n)&(\n(&\n'&(\n&\n#B ! \" A A\n87S5M4P21M/.-,\n+C\n*C\n)C\n(C\n'C\nC\n#B ! \" A A\nV87SL,541P2L11,ML.-,B\n)H\n)*\n(\n'\nC\n#B ! \" A A\n2-74S54P21M/.-,B\n59-2:1S<P/2,P/.<;17:19,8\n)C\n(C\n'C\nC\n#B ! \" A A\n,:9L58L7*S-MH524P21ML.-,H*B\n@?>=8<-,L:;HM;,*4,L.\na b c d e f\n)C\n(D\n(C\nD\nC\n#B ! \" A A\nh\nWP,1V-871PSR14P21M-//+.-,+* \"?!871P>14>=-;7-P8+1<1;;+7.\ng\nsamples. All numerical data are presented as mean ± SD.\n12. In addition, Fig. 4j clearly shows that heart and kidney are significantly labelled by AAM.\nResponse: We would like to clarify that there was always some Cy5 background signal after\nwe stained the tissue sections with DBCO-Cy5. Therefore, the difference between AAM and\nPBS groups, instead of the overall Cy5 signal, indicates the azido labeling efficiency. We also\nanalyzed the average Cy5 fluorescence intensity; negligible differences between AAM and\nPBS groups were observed (Fig. 4k).\nWe have added Fig. 4k and the following text in the revised manuscript: “Confocal imaging of\ntissue sections, after staining with DBCO-Cy5, also showed negligible differences in Cy5\nfluorescence intensity between AAM and PBS groups (Fig. 4j-k).”\nSPPP\n5PPP\n4PPP\nP\n!\" #A\nB\nC '\n#\"(\nF * B\nH\nFA I\n'\"\n-\nKL ' '\nB\nMA\n13. In vivo targeting of DBCO cargo: please comment Figure S6. The differences among day\n0 and day 1; in addition explain while day 5 Cy5 Fl ratio appears to decrease respect day 3.\nResponse: The difference between day 0 and day 1 could be attributed to the release of non-\nspecifically bounded DBCO-Cy5. The Cy5 FI ratio changes between day 5 and day 3 could be\ndue to the death of RBCs over a longer culture time.\n14. Long-term fluorescence imaging: labelled RBC have been administered i.p. and not i.v.,\nplease comment. Is this modality more appropriate for targeting/labelling implanted tumor\ncells?\nResponse: To clarify, we i.p. injected DBCO-Cy5 (instead of labeled RBCs) into mice for the\nfluorescence imaging study. i.p. injection was chosen as it enables the sustained release of\nadministered cargo over an extended time.\n15. MRI imaging of brain blood vessels: Assuming that Gd coupled to RBC is stable were Gd\nends when disappear from circulation? Is this removed in spleen and liver? Does cause any\nkidney toxicity? Please address these comments.\nResponse: It is true that DOTA-Gd could gradually dissociate and release Gd2+ over time. Free\n<;8:298'#H\"'I7\n=!-\n77>\nGd2+ is primarily cleared through the renal clearance, while intact Gd-conjugated RBCs (when\nbecome aged) are expected to be sequestered and cleared by the spleen and liver. In our study,\nwe have not observed any sign of toxicity in major organs including kidneys. Also, mice\nshowed no significant changes in body weight and no sign of adverse effects for over a year,\nsupporting the benign safety profile for the dosage of DBCO-Gd that we used in the study.\n16. In vivo conjugation of drugs to RBCs: DBCO-doxorubicin intravenously administered can\nlabel only about 1% of RBC at 2 hours and is not significant versus the administration of PBS.\nThese results are of very limited interest and doesn’t prove any potential therapeutic benefit.\nResponse: We appreciate the Reviewer’s comment. We agree that the targeting effect of\nDBCO-Dox was not impressive. Nevertheless, we would like to present the representative data\nwe have obtained to showcase the broad applicability of our RBC labeling and targeting\ntechnology, while also telling our readers there is significant room for improvement. Regarding\nthe limited targeting efficiency of DBCO-Dox, the rapid clearance of injected DBCO-Dox and\nthe potential premature release of conjugated Dox from RBCs could be among the reasons.\nFuture studies should optimize the chemical linkages between DBCO and drugs and the overall\nphysiochemical properties of DBCO-drugs.\n17. The same apply to coupling of PE. In this case DBCO-functionalized PE is added ex-vivo\nfor two hours to AAM activated RBC. The same apply to DBCO-insulin detected with rabbit\nanti insulin and Cy5-conjugated goat anti-rabbit secondary antibody on RBC after an ex-vivo\nincubation of two hours. The difference versus mice receiving PBS is not significantly\ndifferent. In vivo experiments with peritoneal administration of DBCO-insulin in AAM treated\nSTR mice show a limited effect only at one time point questioning the benefit of this claimed\ntherapeutic treatment. This should be commented in more details highlighting the limitations\nof the approach.\nResponse: We thank the Reviewer for this comment. We fully agree with the Reviewer\nregarding the limited targeting efficiency of DBCO-cargos. These limitations are exactly what\nwe aim to address in our ongoing and future work, in order to realize the full potential of our\nin vivo RBC labeling technology. We have now added more discussions on these limitations\nin the revised manuscript: “Our in vivo RBC metabolic labeling approach offers a minimally\ninvasive and modular strategy for RBC functionalization, but several limitations warrant\nattention. Differences between murine and human RBCs may impact the overall labeling\nefficiency and cargo retention time, and the potential immunogenicity upon repeated dosing of\ncargos remains to be fully assessed. Also, the amounts of cargos, especially high-molecular-\nweight cargos, that can be conjugated to circulating RBCs are still limited. For example, the\namount of DBCO-DOTA-Gd and DBCO-insulin that could be conjugated to circulating azido-\nlabeled RBCs was relatively low, leading to a modest efficacy. The definite number of azido\ntags per RBC is certainly a main factor, but the physicochemical properties of DBCO-cargos\nand the chemical linkage between DBCO and cargo also require further optimization.\nCompared to existing RBC engineering methods, our approach bypasses the process of\nisolating RBCs, engineering RBCs ex vivo, and infusing the engineered RBCs, but further\noptimization of the labeling and targeting specificity and efficiency is needed to realize the\ntranslational potential.”\nReview #2\nThe study by Yusheng Liu et al describes a novel approach for RBC’s engineering with the\npotential application in imaging and drug delivery. Overall, the study is well designed and\nexecuted, following are a few comments that could further benefit the work:\nResponse: We thank the Reviewer for the positive comments, and addressed the remaining\nissues in the revised manuscript.\n1. RBCs are critical for gas exchange, endothelial functions, platelet reactivity, thrombosis and\nhaemorrhage, binding to pathogens, scavenging chemokines. Furthermore, the elasticity of\nRBC’s is essential for vascular functions specially at the presence of prooxidants. These\nfunctions require through testing to ensure the competence of RBCs after azido tagging and\ncargo incorporation. Tests such as membrane integrity tests (osmotic and Mechanical),\noxidative haemolysis assay, Ektacytometry, and RBC’s indices such as MCV, RDW, MCH\nand MCHC; can augment the safety profile of the proposed strategy.\nResponse: We thank the Reviewer for this valuable comment. We have now supplemented the\nosmotic fragility and mechanical hemolysis analysis of azido-labeled RBCs vs control RBCs,\nby incubating RBCs in varied concentrations of sodium chloride or with varied mechanical\nrotation times. As shown in Fig. S6a-b, negligible differences between azido-labeled RBCs and\ncontrol RBCs (azide-negative RBCs after AAM treatment or untreated RBCs) were observed.\nWe have also analyzed key RBC indices including MCV, MCH, MCHC, and RDW, which\nshowed negligible differences between AAM-treated RBCs and PBS-treated RBCs (Fig. S7).\nThese results further support that azido tagging does not impair RBC membrane function or\nhomeostasis under the conditions tested. Although we were unable to perform ektacytometry\ndue to the lack of available equipment on campus, we have added a discussion of this limitation\nand the importance of comprehensive functional assessment for future translational efforts.\nWe have now added Fig. S6 and Fig. S7 and the following text in the revised manuscript: “We\nalso analyzed the membrane osmotic and mechanical fragility of RBCs collected from AAM-\ntreated mice (both azide-positive and azide-negative fractions) and PBS-treated mice, which\nshowed negligible differences (Fig. S6a-b). RBC indices including RBC counts, Hemoglobin\nconcentration, hematocrit percentages, mean cell volume (MCV), mean corpuscular\nhemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), and red cell\ndistribution width (RDW) (Fig. S7a-g), as well as RBC morphology (Fig. S7h), showed\nnegligible differences between AAM and PBS groups. These data demonstrated that AAM-\nmediated metabolic labeling induced negligible changes in RBC membrane fragility and RBC\nfunctions.”\n'!!\nC!\nB!\nA!\n#!\n!\n!\"! !\"# !\"A !\"B !\"C '\"!\n()*+,*-./-M)*N)2NP/(4N5S7\n;M;:4)9,8N2)NSN\n<<=N5<>M?,@7\n\"!!\n<<=N5<>M?,A7 778.179,:M;S\nBCa '! 778.179,:M<S\n=>?\n&!\nA!\n#!\n!\n! \"# #A B& A' &!\n()*+*,)-.*,/M.12)P4RS\nR,RCB)/M2.A).@.\na b\nFigure S6. In vivo metabolic glycan labeling of RBCs does not affect the osmotic and\nmechanical integrity of RBCs. C57BL/6 mice were i.v. injected with AAM or PBS twice\ndaily for three consecutive days. Four days after the final injection, peripheral blood was\ncollected and RBCs were isolated for functional assessment. (a) Osmotic fragility assay\nshowing percentage of hemolysis as a function of NaCl concentration. (b) Mechanical fragility\nassay measuring hemolysis following rotational stress over time. Data are presented as mean ±\nSD. Hemolysis was calculated as the ratio of supernatant absorbance at 415 nm (hemoglobin)\nto the absorbance at 415 nm obtained from an equal number of RBCs fully lysed with the ACK\nlysis buffer.\n*+\n)\n(\n'\nC\n#B \"A !A\nFigure S7. In vivo metabolic glycan labeling of RBCs does not alter hematological indices\nor morphology of RBCs. C57BL/6 mice were i.v. injected with AAM or PBS twice daily for\nthree consecutive days. Four days after the final injection, peripheral blood was collected for\nhematological analysis. Quantified RBC parameters include (a) RBC count, (b) hemoglobin\nconcentration, (c) hematocrit, (d) mean corpuscular volume (MCV), (e) mean corpuscular\nhemoglobin (MCH), (f) mean corpuscular hemoglobin concentration (MCHC), and (g) red cell\ndistribution width (RDW). (h) Representative RBC morphology assessment for individual\nsamples. All numerical data are presented as mean ± SD.\n2. The main proposed usages mentioned in the studies are, imaging and drug delivery. For\nimaging, the reasoning for having a long-term tagging needs to be justified compared to\nclassical imaging techniques which relies on short acting contrast agents. Similarly for RBCs\nas a drug carrier, more discussion is needed to justify the advantages of this pathway compared\nto using exogenous carriers (e.g. nanoparticles).\nResponse: We appreciate this comment from the Reviewer. We have now added more\ndiscussion in the revised manuscript to justify the promise of long-term RBC tagging: “We\nenvision the RBC tagging and targeting technology is promising for long-term imaging and\nP21M(C*L.-,\",\n)H\n)*\n(\n'\n&\n#B \"A !A\n:987MS54P2L1ML.-,\nH&\n*&\n)&(\n(&\n'&(\n&\n#B \"A !A\n87S5M4P21M/.-,\n+C\n*C\n)C\n(C\n'C\nC\n#B \"A !A\nV87SL,541P2L11,ML.-,B\n)H\n)*\n(\n'\nC\n#B \"A !A\n2-74S54P21M/.-,B\n59-2:1S<P/2,P/.<;17:19,8\n)C\n(C\n'C\nC\n#B ! \" A A\n,:9L58L7*S-MH524P21ML.-,H*B\n@?>=8<-,L:;HM;,*4,L.\na b c d e f\n)C\n(D\n(C\nD\nC\n#B \"A !A\nh\nWP,1V-871PSR14P21M-//+.-,+* \"?!871P>14>=-;7-P8+1<1;;+7.\ng\ncertain drug delivery applications. Conventional contrast agents (either small molecules or\nnanomaterials) are short-acting and are often cleared from the circulation within hours. For\nscenarios when long-term monitoring is needed, e.g., continuous monitoring of disease\nprogression, the necessity for repeated dosing of contrast agents adds to the overall cost,\ncomplexity, and the likelihood to lose track of the dynamics of vascularization in diseased sites.\nOur RBC labeling and targeting approach, instead, enables the circulation of contrast agents in\nthe bloodstream and thus continued monitoring of vasculatures for over 4 weeks. Likewise, for\ndrug delivery, endogenous azido-tagged RBCs could retain the clicked cargos for an extended\ntime than conventional drug delivery vehicles such as nanoparticles and microparticles,\npotentially leading to a reduced dosing frequency of drugs and thus reduced side effects.”\n3. The study demonstrated improved pharmacokinetics for drugs like insulin, however, the\nrelease kinetics of conjugated drugs from RBCs are not thoroughly characterized. This is\ncritical for ensuring controlled and predictable therapeutic outcomes.\nResponse: We thank the Reviewer for this comment. We have now supplemented an in vivo\npharmacokinetic study. Mice were treated with AAM to enable in vivo RBC labeling, followed\nby intraperitoneal injection of DBCO-ester-insulin. The blood concentration of insulin was\nmonitored by ELISA. As shown in Fig. S13f, AAM-treated mice exhibited higher plasma\ninsulin levels than PBS-treated mice at all selected time points between 1-24 h post injection\nof DBCO-ester-insulin, supporting the prolonged blood circulation of drugs via our RBC\nlabeling and targeting approach. However, it is noteworthy that the ester linkage between\nDBCO and insulin is easily degradable under in vivo conditions, and we envision the tunability\nof drug release kinetics with the use of different chemical linkages.\nWe have now added Fig. S13f and the following text in the revised manuscript: “We have also\nmonitored the blood concentration of insulin over an extended time. Mice were treated with\nAAM to enable in vivo RBC labeling, followed by i.p. injection of DBCO-ester-insulin. The\nblood concentration of insulin was measured via ELISA. For all the selected time points\nbetween 1 and 24 h post DBCO-ester-insulin injection, AAM-treated mice exhibited a higher\nplasma insulin level than PBS-treated mice (Fig. S13f), supporting the prolonged blood\ncirculation of drugs via our RBC labeling and targeting approach. However, it is noteworthy\nthat the ester linkage between DBCO and insulin is easily degradable under in vivo conditions,\nand we envision the tunability of drug release kinetics with the use of different chemical\nlinkages.”\nFig. S13f. Blood concentration of insulin at different times post i.p. injection of DBCO-insulin\n(10 IU/kg) in AAM-treated or PBS-treated mice.\nReview #3\nThis manuscript from the Wang lab describes the labeling of red blood cells (RBCs) with azido\nsugars for various potential downstream applications. The authors convincingly show that\nRBCs can be metabolically labelled with ManNAz (termed AAM here) both in vitro and in\nvivo. Additionally, the subsequent bioorthogonal reaction (SPAAC) can be performed in vivo\nas well. All of this confirms data from the Bertozzi lab and others concerning these sugars and\nSPAAC reaction. While the levels of the labeling are modest, the interesting and new feature\nhere is that the azide-sugar persists on the cell surface of RBCs much longer than on other cells\nand tissues, allowing for a level of selectivity based on the timing between AAM and DBCO\ndelivery. Using this feature the authors go on to show that they can detect differences in\nvascularization by fluorescence and image blood vessels by MRI. Finally, they conjugate\npharmaceutical cargos to the RBCs and nicely show that insulin can act longer when appended\nto RBCs. Again, the magnitude of the differences here are mostly small, but I believe this is a\npotentially interesting application of metabolic engineering that is worth further exploration,\nparticularly in terms of cargo circulation and delivery. I am supportive of publication. I do have\ntwo comments that should be addressed first.\nResponse: We thank the Reviewer for the positive comments, we have addressed the remaining\ncomments in the revised manuscript.\nComment #1 - I suggest that the authors are more explicit about achieving RBC selectivity by\nwaiting longer between AAM delivery and subsequent treatment with DBCO. I understand that\nthey talk about the unique retention of azides on RBCs versus other tissues several times in the\npaper. However, I think this could be made more clear for non-experts, so that readers do not\ncome away with the impression that other tissues are not labeled at all.\nResponse: We thank the Reviewer for this considerate advice. We have added now clearly\ndescribed the retention-based selectivity for our RBC labeling approach in the Introduction: “It\nis noteworthy that non-specific azido-sugars used in this study can label both RBCs and non-\nRBCs in vivo. However, due to the non-proliferative property and long life-span of RBCs,\nthe installed azido groups are stable in the bloodstream for over 42 days, nearly the life-span (~45\ndays) of mouse RBCs. In contrast, the azido tags on the membrane of leukocytes in the blood\nand cells in healthy tissues such as liver, spleen, lung, heart, brain, and kidney decay to baseline\nlevels within 3 days.”\nComment #2 - How did the authors detect DOX on cells in Extended Data Figure 9d? Was this\nantibody-based as well. I did not see the details for this experiment.\nResponse: DOX was detected directly by flow cytometry using its intrinsic fluorescent\nproperty (488 nm excitation, 575 nm emission, PE channel). We have added this information\nto the Methods section as follows: “Doxorubicin fluorescence signal in RBCs was detected via\nflow cytometry using the PE channel (488 nm excitation and 575 nm emission)”.\nReview #4\nThe submitted study reports in vivo metabolic glycan labeling of red blood cells (RBCs) using\nsystemically administered azido-sugars. The authors claim that RBCs are tagged with azides\nas cellular surface glycans (glycoproteins and glycolipids). The authors showed the surface\nazido tags on RBCs persist >42 days, whereas tags on leukocytes and other cells rapidly decay.\nThe labeled RBCs can capture DBCO-linked cargos by click chemistry, prolonging a cargo’s\ncirculation from hours to >35 days. As examples, one dose of azido-sugar plus a DBCO-\ngadolinium agent yields months-long brain-vasculature MRI, and linking insulin to tagged\nRBCs extends its blood half-life. In summary, the paper claims a stable in vivo RBC-labeling\nplatform that dramatically extends cargo persistence and enables new imaging and drug-\ndelivery applications.\nResponse: We thank the Reviewer for the nice summary, and have attempted to address the\nremaining comments. Regarding the overall innovation, as an intentional shift from our prior\nwork on rational design of trigger-activatable sugar derivatives for cell-selective labeling\n(which we have to admit is rather difficult and our designed sugar precursors are still far from\nsatisfactory) [Nat Chem Biol 2017, 13, 415-424; Chem Commun 2018, 54, 4878-4881; PNAS\n2023, 120, e2302342120], this work explores the possibility of leveraging the intrinsic\nproperties of RBCs to achieve RBC-selective labeling in vivo. We envision the ability to\nmetabolically tag and target circulating RBCs in vivo, as demonstrated in this study, will benefit\nthe RBC research field and the development of RBC-based diagnostics and therapeutics. Also,\nwhile it seems a ‘lazy’ approach to apply metabolic glycan labeling to different types of cells\nin our lab, we have indeed focused on cells that are historically difficult to engineer in our\nrecent efforts. Nucleus-free RBC without normal protein synthetic machinery is one such type\nof cells that metabolic glycan labeling was deemed unlikely; our work will clear those doubts\nand encourage researchers to explore metabolic labeling and targeting of many other types of\ncells that are difficult to engineer.\n1. While the study demonstrates solid technical execution and offers long-lived labeling of\ncirculating RBCs using azido sugars and click chemistry, it fundamentally builds upon well\nestablished strategies already reported by the same research group. The core methodology,\nsystemic delivery of peracetylated azido-monosaccharides for metabolic labeling and\nsubsequent conjugation of DBCO-bearing molecules via SPAAC, has been applied previously\nto dendritic cells, platelets, and adipocytes. In each case, similar labeling and in vivo targeting\nwere made. The difference in this RBC study from previous is not the chemical mechanism,\nbut merely the target cell type (RBCs) and the extended persistence of the azido label. While\nthe longer half-life of labeled RBCs is notable (∼42 days vs ∼4 days for platelets), this reflects\nmore a known biological property of RBCs than an engineered advance. Thus, the central\nnovelty lies in demonstrating expected behavior in a new context. Moreover, the applications\npresented, such as prolonged imaging or drug delivery, are anticipated outcomes of this\nplatform, not surprising or mechanistically new.\nResponse: We appreciate the Reviewer’s thoughtful comment and happy to know that the\nReviewer is well aware of our prior published work. As the Reviewer is quite familiar with\nmetabolic glycan labeling, we believe the Reviewer may echo on two main challenges of this\nplatform: i) cell-selective labeling and ii) successful labeling of some types of cells that are\nhistorically difficult to engineer and for those successful metabolic glycan labeling were\ndeemed unlikely. For the former challenge, we spent quite some years to design trigger-\nactivatable azido-sugars that can selectively label cells of interest under an external trigger\n(UV, NIR light, ultrasound, etc.) or internal trigger (hydrogen peroxide and different enzymes\nincluding histone deacetylase, cathepsin L, ALDH1A1, etc.). We have made a lot of progress\nover the years, but unfortunately none of those compounds can truly ‘exclusively selectively’\nlabel a specific type of cells. And we have to admit this path has been painful for us and others\nin the field. For the latter, as the Reviewer noticed, we have been working on metabolic labeling\nof platelets, adipocytes, and RBCs, which are all historically challenging to engineer but hold\na lot of scientific, diagnostic, and therapeutic potential.\nThis work on RBC labeling lays out an approach to simultaneously addressing the two grand\nchallenges in this field. On one hand, this is the first report of successful metabolic glycan\nlabeling of RBCs either in vitro or in vivo. As RBCs are nucleus-free and do not possess the\nfull set of protein machinery, their ability to metabolize azido-sugars and express azido-tagged\nglycoproteins and glycolipids on the cell membrane was deemed unlikely, if not impossible, in\nthe field. We anticipate this manuscript will encourage more researchers to test out many other\ntypes of cells they were hesitating before. On the other hand, by leveraging the unique\nproperties of RBCs, we show that unmodified azido-sugars (i.e., without any complexed\ntrigger-responsive moiety) accessible to every laboratory can achieve a certain level of labeling\nselectivity towards the circulating RBCs in the body. This is not a trivial advance for the sake\nof RBC labeling/targeting itself and also an alternative strategy to achieve cell-selective\nmetabolic labeling.\n2. Although the study convincingly demonstrates that azido groups persist on the surface of\ncirculating RBCs for over 42 days, it does not adequately establish that this labeling strategy\nis selective enough for practical applications in targeted imaging or drug delivery. The authors\nargue that azide tags decay rapidly in leukocytes and other nucleated blood cells within 3 days,\nthereby enabling selective RBC targeting using DBCO-functionalized molecules if\nadministered after this window. However, this argument overlooks a more critical issue:\nwhether normal tissues and vasculature are also being labeled and whether that labeling persists\nto a degree that compromises selectivity. In particular, Figure 5i-j shows detectable\nfluorescence signals in normal tissues (liver, spleen, etc.) after DBCO-Cy5 injection, even in\nthe AAM-treated group. Although the signal is lower than in RBCs, it is not negligible, and\nthere is no quantification relative to background or functional implications of this residual\nlabeling. The authors use a “wait 3 days post AAM” strategy to reduce leukocyte labeling, but\nit remains unclear whether this delay also fully eliminates labeling in non-blood tissues. The\nWestern blot and confocal data in Fig. 4i-j are helpful but limited, they do not resolve whether\nlow-level but functionally significant tagging persists.\nResponse: We appreciate this another great comment from the Reviewer. We admit one key\nlimitation of our reported RBC labeling and targeting approach is the lack of exclusive\nselectivity, which is and will continue to be a constant issue for any metabolic glycan labeling\nand targeting work. As we aimed to avoid the sophisticated design of enzyme-activatable\nazido-sugars and instead used the non-selective azido-sugars (AAM or AAGal), the off-target\nlabeling of other cells in the blood and tissues is not surprising. As the Reviewer has noticed,\nwe have shared/presented all the off-labeling labeling data in the manuscript, as part of our\nintention to remind our readers that the labeling of RBCs is not exclusively selective. It seems\nto us that the Reviewer is looking for something that can absolutely selectively label cells of\ninterest, which is of course our and others’ ultimate goal but still has a long way to go. For now,\nany advance in achieving ‘more selective’ labeling and targeting of cells of interest should be\nencouraged and is important in this field.\nRegarding Fig. 4j, we have now added the semi-quantitative data of Cy5 fluorescence intensity\nin different tissues as Fig. 4k, which shows negligible differences between AAM and PBS\ngroups in brain, heart, lung, liver, spleen, and kidney. Both the ex vivo staining data and in\nvivo DBCO-Cy5 conjugation data show that the azido labels in healthy tissues drops to near-\nbackground levels at 3 days or later post AAM injection. Again, we do not want to hide the\nfact that there would always be some levels of off-target labeling. From the pharmacology and\ndrug development perspective, off-target labeling or binding constantly exists but should not\ndiscourage the exploration of drugs or strategies that can preferentially effect on the target cell.\nFig. 4k. Normalized Cy5 fluorescence intensity of tissues in (j).\n3. Furthermore, the authors‘ own prior studies on metabolic labeling of platelets, adipocytes,\nand dendritic cells reported relatively broad azido labeling, including some tissue types. The\ncurrent claim that RBCs uniquely retain azido tags while all other tissues rapidly clear them\nseems inconsistent with those earlier findings, suggesting either differing experimental\nstandards or unexplored biological variability. This inconsistency undermines the claim of\nRBC-specific labeling and suggests that more rigorous, quantitative tissue-distribution studies\nare needed.\nResponse: We appreciate the Reviewer’s mention of our previous work. Indeed, we have\nconsistently reported that the density of azido tags on dendritic cells, platelets, and adipocytes\ndecays rapidly to a baseline level in 2-4 days in our previous publications and in this\nmanuscript, which is exactly the reason why the long-term labeling of RBCs is special.\n4. Overall, The study is technically well-conducted and shows promise for translational\napplications, but it does not introduce a sufficiently novel concept or mechanism to meet the\npublication standards of Nature Communications. The core advances are too directly on a\nsequence of prior works from the same group using near-identical strategies. There is no\ndemonstration of fundamentally new biological insight, molecular mechanism, or chemical\nmethodology that would justify publication in a journal prioritizing broad conceptual advances.\nIn addition, the study does not sufficiently demonstrate RBC-specific labeling or targeting, and\nseveral results raise the possibility of non-negligible off-target effects in normal tissues. This\nundermines the claimed value of the system for selective imaging or drug delivery.\nAdditionally, the conclusions appear partially inconsistent with earlier work from the same\ngroup on metabolic labeling of other cell types. Without stronger and more systematic evidence\nof selectivity, this work does not meet the robustness or clarity needed for publication in Nature\nCommunications.\nResponse: We believe these comments have been covered above. We would like to thank the\nReviewer again for their deep knowledge of our prior work and excellent comments on the\nlabeling selectivity of our approach. Novel chemistry design of unnatural sugars for cell-\nselective labeling is one direction we have pursued (probably pioneered if the Reviewer agrees;\n[Nat Chem Biol 2017, 13, 415-424; Chem Commun 2018, 54, 4878-4881; PNAS 2023, 120,\ne2302342120]) for years, and we are happy to share with the community that conventional\nazido-sugars without any complexed chemistry design could also achieve a high level of\nlabeling selectivity towards certain types of cells by leveraging the unique biological properties\nof cells. While the RBC labeling approach in this work is not exclusively selective, as expected,\nit provides a promising tool for researchers to explore improved RBC-based diagnostics and\ntherapeutics.\nPoint-by-point reply to Reviewers' comments\n(All responses were colored in blue; all changes in the manuscript were highlighted in\nyellow)\nReviewer #1:\nDuring the revision process the Authors have addressed many critical points and provided new\ndata and explaining some of the limits of this approach. This is appreciable, although not\nconclusive, and support acceptance from the point of view of this Reviewer.\nResponse: We thank the Reviewer for the positive feedback and comments, and have addressed\nthe remaining comments in the revised manuscript.\nMain section: The Authors have amended the previous version of the paper that now read\n“…Significant progress has indeed been made to address these issues and bring RBC-based\ndrug delivery systems to clinical trials.20,21”. In addition “While direct encapsulation of\ncargos into RBCs has been extensively explored, most approaches rely on ex vivo engineering\nof RBCs,48,49 which inevitably inherits the common issues in RBC stability, infections, costs\nduring the isolation, storage, ex vivo manufacturing, and infusion processes.” I respectfully\ndisagree. The technology nowadays has been greatly improved and was not only taken into\nphase 3 clinical trial but the trial has also documented the safety of this approach in hundreds\nof patients (see Ref 21). This sentence should be amended. Ref 20 is not correct since reports\nthe title of Ref. 21\nResponse: We appreciate this comment from the Reviewer, and agree with the Reviewer that\nthe past decades has witnessed significant progress in the development of ex vivo engineered\nRBC products. We have now deleted “While direct encapsulation of cargos into RBCs has been\nextensively explored, most approaches rely on ex vivo engineering of RBCs, which inevitably\ninherits the common issues in RBC stability, infections, costs during the isolation, storage, ex\nvivo manufacturing, and infusion processes”. Deleting this sentence does not affect the flow of\nthe manuscript at all and avoids the confusion mentioned by the Reviewer. We have also\ndeleted Ref. 20 which was cited wrongly.\nImmunogenicity: New sentence “We are aware of the concern on the potential immunogenicity\nof cargos being conjugated to circulating RBCs. In separate studies, we attempted to conjugate\nDBCO-functionalized 2,4-dinitrophenylated Hemocyanin, Keyhole Limpet (DNP-KLH) and\nDBCO-ovalbumin (OVA), two representative antigens, to RBCs in vivo and monitor the\nantibody titers over time. As a result, the in vivo conjugation of DNP-KLH and OVA antigens\nfailed to exert any enhancement on the antibody responses compared to the control groups (Fig.\nS9), which lessens the concern of potent immunogenicity of RBC-conjugated cargos in our\nmetabolic labeling approach.” The new experiment id done by administering 0.1 mg of DNP-\nOVA or DNP-KLP that likely will partially be bound to activated RBC while partially will\nremains free in circulation. I’m encouraging the Authors to provide an estimate the percentage\nof the administered antigen that is RBC conjugated vs. the total administered.\nResponse: We agree with the Reviewer that only a small fraction of injected DBCO-antigens\nwould be captured by azido-labeled RBCs. Per our estimation, likely < 1% of injected DBCO-\nOVA and <10% of injected DBCO-DNP-KLH would be conjugated to circulating RBCs [~10%\nof azido-labeled RBCs, 1×107 RBC/µL, and 2 mL of circulating blood in mice yield ~2×109\nazido-labeled RBCs. Assuming ~103-104 azido groups per RBC based on our prior studies,\ncirculating RBCs have a total number of 2×1012 - 2×1013 (i.e., 3.3×10-12 - 3.3×10-11 mole) azido\ngroups. With 100% consumption of azido groups, 0.15-1.5 µg DBCO-OVA (45 kDa) or 1.2-12\nµg DBCO-DNP-KLH (350 kDa) could be theoretically conjugated. These correspond to 0.15-\n1.5% I.D. for DBCO-OVA and 1.2-12% I.D. for DBCO-DNP-KLH].\nWe have added the following text in the revised manuscript: “We estimate < 1-10% of\nadministered DBCO-protein could be conjugated to circulating RBCs, and the in vivo RBC\nlabeling and targeting conditions require further optimization to increase the amount of\nconjugated cargos.”"}]

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