- Open Access
Probing the subcutaneous absorption of a PEGylated FUD peptide nanomedicine via in vivo fluorescence imaging
© The Author(s) 2019
- Received: 22 April 2019
- Accepted: 12 June 2019
- Published: 8 July 2019
The Functional Upstream Domain (FUD) peptide is a potent inhibitor of fibronectin assembly and a therapeutic candidate for disorders linked with hyperdeposition of fibronectin-modulated ECM proteins. Most recently, experiments involving subcutaneous (s.c.) administration of a PEGylated FUD (PEG-FUD) of 27.5 kDa molecular weight yielded a significant reduction of fibronectin and collagen deposition in a murine model of renal fibrosis. The benefits of FUD PEGylation need to be studied to unlock the full potential of the PEG-FUD platform. This work studies the impact of PEGylating the FUD peptide with differently sized PEG on its absorption from the site of injection following s.c. delivery using non-invasive in vivo fluorescence imaging. The FUD and mFUD (control) peptides and their 10 kDa, 20 kDa, and 40 kDa PEG conjugates were labeled with the sulfo-Cy5 fluorophore. Isothermal titration calorimetry (ITC) and confocal fluorescence microscopy experiments verified FUD and PEG-FUD fibronectin binding activity preservation following sulfo-Cy5 labeling. Fluorescence in vivo imaging experiments revealed a linear relationship between the absorption apparent half-life (t1/2) and the MW of FUD, mFUD, and their PEG conjugates. Detected drug signal in the kidney and bladder regions of mice suggests that smaller peptides of both the FUD and mFUD series enter the kidney earlier and in higher amounts than their larger PEG conjugates. This work highlights an important delayed dose absorption enhancement that MW modification via PEGylation can contribute to a drug when combined with the subcutaneous route of delivery.
- Renal fibrosis
- Subcutaneous drug absorption
Renal fibrosis presents a significant clinical challenge that demands development of novel and effective therapeutics. The current standard of care for renal fibrosis involves administration of angiotensin-converting enzyme inhibitors (ACEI) or type 1 angiotensin II receptor blockers (ARBs) that can slow the decline in kidney function but do not revert the morphological damage done to the kidneys . Chronic inflammation and hyperdeposition of collagen are hallmark features of fibrosis. Because of its involvement in both processes [2, 3], fibronectin (FN) has been implicated as a possible therapeutic target for the treatment of fibrotic disorders. Interruption of FN fibril formation and thus ECM deposition can have the downstream effect of modulating attachment of lymphocytes and deposition of collagen and other ECM proteins at the site of injury. The pioneering work of Mosher et al.  showed that the terminal 70K region of FN is critical to its ECM assembly. This information was used to engineer a large 6 kDa peptide, the Functional Upstream Domain (FUD) peptide also known as the pUR4 peptide, to have a low nanomolar affinity for the 70K region of FN and thus to have potent FN matrix assembly inhibition activity [5–7]. The FUD peptide was successfully applied in murine models of liver fibrosis, coronary artery disease, and heart failure to reduce the fibrotic burden of each disease [8–10]. The Kwon lab sought to increase the efficacy of this novel therapeutic through modification of FUD at the N-terminus with polyethylene glycol (PEG) of 10 kDa, 20 kDa, and 40 kDa MW, increasing the effective hydrodynamic size of FUD and thereby improving its delivery with a nanotechnology approach. All three variants of this PEG-FUD displayed preserved binding affinity (Kd) and in vitro FN matrix assembly inhibitory potency of the native FUD peptide . These in vitro and biophysical results complement later successful application of PEG-FUD in a murine model of renal fibrosis.
Efficacy evaluation of a singly 20 kDa PEGylated FUD peptide in the unilateral ureteral obstruction (UUO) murine model of renal fibrosis revealed significant amelioration of renal fibrosis morphological features . Administration of the peptide for seven consecutive days yielded a reduction in levels of fibronectin (~ 70%), collagens I and III (~ 60%), and CD45-expressing cells (~ 50%) in the kidney. Interestingly, PEG-FUD was twice as efficacious in reducing FN content of the diseased kidneys as unmodified FUD. This efficacy improvement is perhaps due to an enhanced plasma exposure and thus therapeutic window that is typical of PEGylated drugs. It is well understood that PEGylation using a sufficiently large PEG moiety can result in a reduction of renal clearance and proteolytic degradation of a drug [13–15]. This is true because a PEG moiety with a hydrodynamic radius of sufficiently large size can significantly reduce the drug’s renal sieving coefficient and can also sterically hinder the interaction of proteolytic enzymes with the drug. Furthermore, PEGylation can provide additional therapeutic enhancements if coupled with the subcutaneous (s.c.) delivery route. Molecular weight (MW) modulation via PEGylation can reduce the rate of absorption and consequently the rate of systemic release of the drug, further enhancing the drug’s therapeutic window. Because the s.c. route is common and thus therapeutically relevant for the delivery of nanomedicines, understanding this aspect of PEGylated FUD is important to informing future development of this therapeutic platform as well as that of other nanomedicines.
Although much remains to be understood about the mechanism of this effect, the relationship between the rate of protein s.c. absorption and MW has been studied in the past in several animal models [16–18]. Peptides and proteins that are small (< 16–20 kDa) are known to enter circulation from the s.c. site primarily through blood capillary diffusion. Larger biomolecules occupying a greater volume like nanobodies and monoclonal antibodies are excluded from this pathway and instead must traverse through the interstitium before entering the lymphatic system, where they cross lymph capillaries into lymph nodes before ultimately entering blood circulation. As a result, the time to maximum serum concentration (Tmax) of monoclonal antibodies administered subcutaneously is around 3–8 days in humans . The work of Kaminskas et al.  explored this concept using interferon (IFN, 19 kDa) and its PEGylated variants, IFN-PEG12 (31 kDa) and IFN-PEG40 (60 kDa). It was found that while IFN shows poor uptake into the lymph (< 1%), approximately 20% and 21% of the injected IFN-PEG12 and IFN-PEG40 dose, respectively, was recovered in the thoracic lymph following s.c. administration. Furthermore, the Tmax of each drug increased with drug MW, suggesting an inverse relationship between drug s.c. absorption and MW. These results thus support the model of increasing drug MW and thus hydrodynamic radius redirecting the pathway of drug transit into systemic circulation and thus reducing the rate of s.c. absorption.
In contrast to other work tracking a series of different proteins of different MW in the plasma or the lymph [20–22], this work explores the behavior of a parent peptide and its PEG conjugates through direct observation of the remaining quantities of the drug at the site of injection as a function of MW. To accomplish this task, a series of three PEG-FUD conjugates have been synthesized. The FUD and 10 kDa PEG-FUD peptides have a MW (6 kDa, 17 kDa) that lies below the 16–20 kDa capillary diffusion pathway cutoff whereas the 20 kDa and 40 kDa PEG-FUD have a MW (27.5 kDa, 49.5 kDa) that matches the lymphatic flow pathway. The FUD peptide and all three of its PEG conjugates have additionally been attached with a sulfo-Cy5 label to facilitate direct observation of drug levels via in vivo fluorescence imaging. Furthermore, the work was repeated using an analogous peptide with no activity for fibronectin, the mutated FUD peptide termed mFUD. Together, the FUD and mFUD series will provide two case studies demonstrating the effect of size modification via PEGylation on the s.c. absorption of a a nanotherapeutic. This work will illustrate a powerful therapeutic property of PEGylation when coupled with the s.c. delivery route, informing future development of PEG-FUD and converging it with development of nanomedicines in general.
2.1 Generation of FUD, mFUD, and 10–40 kDa PEG conjugates
FUD and mFUD were generated using a recombinant peptide synthesis protocol with His-tag removal modifications  reported previously. Their PEG conjugates were generated using reductive amination chemistry as described in previous work . This task was accomplished using methoxy-PEG-aldehyde of the 10 kDa, 20 kDa, and 40 kDa size purchased from NOF Corporation (Kawasaki, Japan). The concentration of FUD, mFUD, and their PEG conjugates was obtained from 280 nm absorbance measurements using ε = 2980 L mol−1 cm−1 and ε = 4470 L mol−1 cm−1 for FUD and mFUD, respectively.
2.2 Generation of sulfo-Cy5 labeled FUD, mFUD, and 10–40 kDa PEG conjugates
Each peptide was labeled with the sulfo-Cy5 fluorophore by incubating a 2 mg/mL solution of the peptide presented in 20 mM Tris buffer (pH 8) with 1 eq of 10 mg/mL Sulfo-Cy5-NHS (Lumiprobe) stock solution originally dissolved in DMSO. The reaction proceeded at room temperature for 2 h under stirring conditions. The reaction mixture was then dialyzed ON using 20 mM Tris (pH 8) and a 3000 MWCO dialysis membrane to remove the unreacted label. The reaction products were then purified using ion-exchange chromatography by loading the solution onto a HiTrap Q HP anion exchange column (GE Healthcare Life Sciences, USA) initially equilibrated with Buffer A (20 mM Tris, pH 8.0). Upon sample injection, the column was washed with 2 CVs of Buffer A and then the sample was eluted with a 10 CV 100% gradient of Buffer B (1 M NaCl in 20 mM Tris, pH 8.0). The fraction containing singly labeled drug was collected and snap frozen. The concentration of the labeled drug was determined using absorbance measurement at 646 nm and the extinction coefficient of sulfo-Cy5 at that wavelength, ε = 271000 L mol−1 cm−1.
2.3 RP-HPLC analysis of sulfo-Cy5 conjugates
Analysis of purified FUD, mFUD, and their 10–40 kDa PEG conjugates was facilitated by a Zorbax SB-C8 4.6 × 75 mm column with a 3.5 μm pore size (Agilent) connected to a Prominence UPLC system (Shimadzu). A gradient of water and acetonitrile both containing 0.1% formic acid at a flow rate of 1 mL/min was used to elute the sample. A fluorescence detector set to the excitation and emission wavelength of sulfo-Cy5 (ex: 646 nm, em: 662 nm) was used to detect the labeled peptides.
2.4 Isothermal titration calorimetry (ITC) of sulfo-Cy5 conjugates
Isothermal Titration Calorimetry experiments of FUD, FUD-Cy5, and 20 kDa PEG-FUD-Cy5 were performed using a VP-ITC (MicroCal, LLC) microcalorimeter with a cell volume of 2.2 mL. Both the peptides and the FN were inserted into separate 3000 MWCO dialysis bags and were dialyzed ON simultaneously into the same 2L Phosphate Buffered Saline (PBS, pH 7.4) solution before each experiment. A typical ITC experiment involved titration of a 35 μM peptide solution into a cell filled with 1.4 mL of 2.7 μM human plasma fibronectin (MilliporeSigma) at a temperature of 25 °C. A total of 39 injections (1 × 1, 4 × 4, and 34 × 8 μL) were delivered in 120 s intervals. The first data point was routinely discarded and a peptide into PBS control experiment was subtracted from each run to account for the peptide heat of dilution. Data were fit using a one set of sites model Lavenberg-Marquardt nonlinear regression in Origin 7.0.
2.5 Confocal fluorescence microscopy of sulfo-Cy5 labeled peptides
The binding of sulfo-Cy5 labeled peptides and their PEG conjugates to developed extracellular FN networks of dermal fibroblast was observed using confocal fluorescence microscopy. An AH1F fibroblast suspension containing 60,000 cells in 2% fetal bovine serum (FBS) + Dulbecco’s Modified Eagle’s Medium (DMEM) was added to a 35 mm Glass bottom dish with 20 mm micro-well (Cellvis). The cells were incubated for 2 h at 37 °C with 5% CO2 to facilitate spreading and adhesion of cells. Following incubation, 100 μL of either human plasma FN or Alexa Fluor 488 (ThermoFisher) labeled human plasma FN (A488-FN) was then added to each well for a final concentration of 11 μg/mL. The cells were then incubated for 24 h at 37C to facilitate FN matrix formation. The A488-FN was generated from the same stock of FN as the experiment’s using the manufacturer’s protocol for the NHS ester. On the next day, the liquid contents of the well were removed and replaced with 100 μL of a 500 nM peptide treatment. After a 30 min RT incubation, the cells were washed with Hank’s Balanced Salt Solution (HBSS) containing Ca2+ and Mg2+ three times and then incubated for 5 min with 100 μL of 5 ug/mL Hoechst 33,342 nucleic acid stain (ThermoFisher) in 2% FBS + DMEM. The cells were again washed three times with HBSS containing Ca2+ and Mg2+ and presented for imaging. Images were captured using an Olympus FV1000 laser scanning confocal microscope and optimized using FV10-ASW software. The following channels were used for image acquisition: Hoechst—405 nm laser; Alexa 488–488 nm laser; Cy5—635 nm laser. The AH1F cells used in this study are human foreskin fibroblasts that have been described previously  and used to demonstrate incorporation of Alexa488-fibronectin in the extracellular matrix .
2.6 Fluorescence imaging of FUD, mFUD, and 10–40 kDa PEG conjugates subcutaneous absorption
Female nude athymic 8–10 week old, 20–23 g mice were purchased from Envigo (Madison, WI) and were housed in the Wisconsin Institutes for Medical Research (WIMR) animal facility at the University of Wisconsin-Madison with ad libitum access to food and water. Nude mice were chosen as the model animal because their lack of hair simplifies fluorescence imaging experiments. Animals were maintained in humidity and temperature-controlled rooms under 12 h light/dark cycles. All work was conducted under protocol M005844, reviewed and approved by the University of Wisconsin-Madison Institutional Animal Care and Use Committee. A standard drug dose contained 36.2 nmol (12.5 mg/kg of FUD equivalents) of FUD, mFUD, or 10–40 kDa PEG conjugate and 0.552 nmol of its corresponding sulfo-Cy5 conjugate in 100 μL. In other words, each drug dose was spiked with 1.5% of the sulfo-Cy5 labeled drug for a final sulfo-Cy5 labeled drug concentration of 5.52 μM. This concentration was chosen because of previously performed pilot studies and well-plate experiments showing no significant fluorophore signal saturation or quenching activity in that concentration range. The total amount of drug present in each dose was chosen to parallel previous work in the murine UUO renal fibrosis model . A drug dose stock solution was prepared by mixing an appropriate amount of unlabeled and labeled drug in a conical vial. The solution was dialyzed overnight into PBS, pH 7.4 using a 3000 MWCO dialysis membrane. The drug dose stock solution was then reduced in volume to the appropriate drug concentration using a 3000 MWCO Amicon Ultra-4 centrifugal unit. All dose concentrations were verified before injection. All drug doses were filtered using a sterile 0.2 μm syringe filter prior to injection.
The FSC% values extracted from each drug series were modeled using nonlinear regression in GraphPad Prism (8.0.2, GraphPad Software, San Diego, California, USA). To accomplish this task, the one phase decay function with a 0 ≤ terminal plateau constraint was applied to the terminal phase of each data series. This function’s fitting model is: FSC = (FSC,0 − Plateau) · exp (−k · t) + Plateau, where FSC,0 is the dose fraction present at the site of injection at time = 0, Plateau is the value of the signal’s terminal asymptote, and k is the rate constant for the loss of drug from the s.c. site. Because the larger drugs demonstrated a signal reduction lag in the initial time points, the model was applied to the terminal time points of 0–48 h, 1–48 h, 3–48 h, and 6–48 h for FUD and 10 kDa, 20 kDa, and 40 kDa PEG conjugates, respectively. The apparent half-life (t1/2) of the drug absorption indicating 50% loss of drug from the s.c. site was calculated from this fitting model using the condition of FSC = 50. The GraphPad Prism software was used to analyze significant differences among FSC values of FUD and mFUD peptides in one MW group as well as a group containing peptides and their immediately larger MW partners. The Student’s t-test with the Holm-Sidak method multiple comparisons correction was used to accomplish this task. Probability (p) ≤ 0.05 was considered to be significant.
3.1 Synthesizing and purifying peptides and their sulfo-Cy5 conjugates
The FUD, mFUD, and 10–40 kDa PEG Conjugate peptides were synthesized and labeled with sulfo-Cy5 to support later in vivo fluorescence imaging experiments.
3.1.1 Sulfo-Cy5 conjugate synthesis
The sulfo-Cy5 labeled native and PEGylated peptides were successfully synthesized via coupling of the NHS reactive group of the sulfo-Cy5 fluorophore and the primary amines of the Lysine residues and the N-terminus of the peptides. This reaction chemistry is nonspecific at the pH 8 reaction conditions used in this synthesis. Following incubation of the drug and the label, the intensely blue solution was dialyzed using pH 8 Tris. The previously clear dialysate gained a slight blue color, indicating that unreacted dye had passed from the reaction products into the dialysate. This observation suggests that some unlabeled peptide remained in solution as the sulfo-Cy5 and the drug were added in equimolar quantities. Following dialysis, the synthesis products were purified using ion-exchange chromatography (IEX).
3.1.2 Sulfo-Cy5 conjugate chromatographic characterization
3.2 Probing the fibronectin binding interaction with FUD-Cy5 and 20 kDa PEG-FUD-Cy5
The interaction of FUD-Cy5 and 20 kDa PEG-FUD-Cy5 with human plasma FN was studied to determine whether its strength is reduced by chemical derivatization of the drug with sulfo-Cy5.
3.2.1 Isothermal titration calorimetry
Extracted isothermal titration calorimetry (ITC) experiment binding parameters
1.48 (± 0.01)
4.7 (± 0.1)
− 11.37 (± 0.02)
− 35.8 (± 0.2)
− 82.1 (± 0.5)
20 K PEG-FUD-Cy5:FN
1.50 (± 0.01)
13. (± 2)
− 10.8 (± 0.1)
− 35. (± 1)
− 82. (± 4)
1.47 (± 0.05)
4.3 (± 0.1)
− 11.40 (± 0.02)
− 33. (± 2)
− 71. (± 6)
1.59 (± 0.06)
6. (± 3)
− 11.4 (± 0.3)
− 31. (± 1)
− 65. (± 3)
20 K PEG-FUD:FN
1.63 (± 0.07)
10. (± 2)
− 10. (± 1)
− 30. (± 1)
− 66. (± 7)
The findings of this ITC study reveal an interesting feature of the FUD peptide that has powerful consequences on the peptide’s ability to be used as an imaging agent. The lack of change in FUD-FN binding affinity upon sulfo-Cy5 labeling is perhaps due to the nature of the interaction between FUD and FN. It involves a cooperative binding of FUD residues located along an extensive region of the peptide with six regions of fibronectin, together contributing to a tight, nanomolar avidity . It is possible that the six possible sulfo-Cy5 points of conjugation (five Lysine residues and the N-terminus) reside in FUD domains that are not critical to this interaction, leading to a lack of binding affinity reduction. The sulfo-Cy5 N-hydroxysuccinimide ester (NHS) reactive group is also separated from the fluorophore’s body by a C6 tail, possibly also spatially contributing to this effect. Altogether, the lack of binding affinity reduction suggests that sulfo-Cy5 conjugation does not significantly affect each peptide’s ability to bind to FN, that fluorophore-labeled FUD and its PEGylated analogs will retain their FN fibrillogenesis inhibitory potency, and consequently that the labeled peptides will display a similar in vivo therapeutic action. This suggestion is further supported by in vitro fluorescence microscopy experiments of sulfo-Cy5 labeled peptides binding to assembled exogenous human plasma FN matrix.
3.2.2 Fluorescence microscopy
3.3 Probing drug s.c. absorption via in vivo fluorescence imaging
The in vivo s.c. absorption and kidney localization of FUD, mFUD, and their PEG conjugates following s.c. injection was studied using IVIS fluorescence imaging.
3.3.1 Subcutaneous drug absorption
Extracted drug s.c. absorption parameters
FSC, 24 h (%)
0.6 (± 0.1)
1.47 (± 0.09)
4. (± 1)
10. (± 3)
0.00 (± 0.04)
0.43 (± 0.05)
1.9 (± 0.3)
7. (± 2)
3.3.2 Peptide penetration into the kidney and the bladder
The observation that FUD and mFUD peptides with increased MW enter the kidney in smaller abundance is consistent with classical theory of glomerular filtration. Although the relationship is complicated by acidity, charge, and geometry of the filtrate [26, 27], it is understood that there exists an inverse relationship between the hydrodynamic volume of a molecule and its ability to participate in glomerular filtration and thus renal elimination. Studies using a variety of model molecules have demonstrated this effect. Most relevantly, a study found renal clearance of an i.v. bolus infused PEG series (6–190 kDa) in a mouse model rapidly declining with MW of 20 kDa or greater . The MW of peptides used in this study is 6 kDa, 16.9 kDa, 27.4 kDa, 49.6 kDa for the parent FUD or mFUD peptides and their PEG conjugates of increasing mass, respectively. The 20 kDa threshold supports this work’s observations as the MW of 10 kDa and 20 kDa PEG-FUD peptides is just smaller and just greater than this threshold. Consequently, the 10 kDa PEG-FUD can be clearly detected in the kidneys while the 20 kDa PEG-FUD is poorly resolved. The 40 kDa PEG-FUD is detected most poorly of the entire set. It is unclear whether the detected signal is of intact peptides or sulfo-Cy5 containing peptide fragments. It is possible that PEGylation also reduces the peptide’s proteolytic degradation in some proportion with MW, amplifying the observed difference in kidney intensity between the peptides of different MW. Whether via renal elimination or other pathways, it is evident that PEGylation has a clear protective impact, decreasing the elimination and increasing the bioavailability of the FUD and mFUD conjugates.
In this work, we demonstrate that increasing the MW of the FUD peptide through PEGylation reduces its absorption from the site of injection following s.c. administration. The drug’s absorption closely follows a linear inverse relationship with respect to MW, where the lower MW peptide enters circulation faster. This task was accomplished using a sulfo-Cy5 peptide labeling methodology combined with non-invasive in vivo fluorescence imaging. These findings carry exciting implications for the field of fibrosis research, and specifically renal fibrosis research, by revealing a path towards heightened therapeutic accessibility of the parent FUD peptide. Fibronectin inhibition is a possible therapeutic strategy that has already yielded successful results in murine models of liver fibrosis, renal fibrosis, coronary artery disease, and heart failure [8–10, 12]. Understanding that the therapeutic window enhancements provided by PEGylation (i.e., reduction of renal clearance and proteolytic degradation) can be further compounded by delivering the size-optimized PEGylated drug subcutaneously and thereby delaying its absorption and systemic release opens a window of opportunities for therapeutic evaluation of PEG-FUD in models of other pathologies. Idiopathic pulmonary fibrosis is one such pathology that has a high impact, has inadequate standard treatment, and whose progression is dependent on fibronectin activity [29–31]. Increasing the therapeutic window of FUD via PEGylation can also increase its overall therapeutic relevance by reducing the frequency with which the drug would need to be injected by the patient. If the drug is released into systemic circulation more slowly and its plasma levels are maintained to a sufficient level, fewer injections are necessary. An increased therapeutic relevance increases the likelihood that a PEG-FUD therapy is successfully translated from murine models into the clinic.
This work’s insight into PEGylated FUD s.c. delivery intersects with work describing s.c. delivery of other large nanomedicines like drug-conjugated dendrimers and monoclonal antibodies (mAbs). Previous research shows that larger dendrimers display delayed lymphatic drainage, suggesting a MW dependent rate of lymphatic transport . Monoclonal antibodies also consistently release from the s.c. injection site over the course of several days and thus more slowly than smaller drugs . Studying the s.c. absorption of the PEG-FUD and PEG-mFUD model system is generalizable to other nanomedicines and thus complements previous research describing them. As this work’s results are restricted to the murine model, much work remains ahead. There exist significant knowledge gaps in our understanding of the full complexity of macromolecule subcutaneous delivery . It is known that the species-specific, subject-specific, and ECM microenvironment-specific characteristics can have a profound effect on the rate of absorption of a drug [16–18]. The convergence of this work with previous research pertaining to these other nanomedicines will help inform future research supporting nanomedicine clinical development.
This work’s methodology also functions as a case study demonstrating an exciting potential function of the PEG-FUD platform: an imaging agent. In this work, the FUD and PEG-FUD peptides were labeled with sulfo-Cy5 via peptide primary amine (-NH2) functionality and the sulfo-Cy5 N-hydroxysuccinimide ester (NHS) functionality. Retention of low nanomolar binding affinity for FN following this process suggests that the drug-label conjugate retains its potent fibrillogenesis inhibitory activity in addition to gaining imaging agent properties, and thus can act as a theragnostic agent. A single dose can act as both a fibrosis therapeutic and an imaging agent for use in localizing regions of injury or evaluating disease progression. The NHS functionality is ubiquitous to other labels and is easily accessible, making the labeled PEG-FUD platform generalizable to other technologies as well. One specific example of this technique’s application includes treatment, visualization, and staging the progression of pulmonary fibrosis . The labeled drug or a combination of labeled and unlabeled drug will likely have both therapeutic and imaging action, allowing both disease treatment and visualization and quantification of the FN rich tissue present in the fibrotic lung. A difference in drug signal over time and thus fibronectin content reduction can then be used as a therapeutic endpoint, allowing both treatment and diagnosis of pulmonary fibrosis.
Conclusively, this work presents two important aspects of the PEG-FUD platform that are interesting to explore in the future. The absorption of the PEGylated drug from the s.c. site of administration should be understood in other animal models to bring the drug closer to the clinic. The diagnostic aspect of PEG-FUD should be studied using other tracer labels to probe the drug’s potential as a diagnostic tool for fibronectin-linked pathology applications. We enthusiastically recommend PEG-FUD as a candidate for study in these two areas.
Authors thank Molly Pellitteri Hahn and Cameron Scarlett at the UW-Madison School of Pharmacy Analytical Instrumentation Center (AIC) for their support in LC–MS experiments and Justin Jeffrey at the University of Wisconsin Carbone Cancer Center (UWCCC) Small Animals Imaging and Radiotherapy Facility (SAIRF) for his support in fluorescence imaging experiments. We thank the staff of the UWCCC Biostatistics Shared Resource (BSR) for their valuable contributions to this research.
The contributions made to this work from the University of Wisconsin Carbone Cancer Center (UWCCC) Biostatistics Shared Resource (BSR) are supported by Cancer Center Support Grant P30 CA014520.
PWZ designed and performed all major experiments. PWZ wrote the manuscript. IT and LR contributed to fluorescence imaging experiments. All authors read and approved the final manuscript.
GSK and PZ are among co-inventors of a filed patent (filing date: Nov 12th, 2018) pertaining to clinical applications of PEG-FUD, including as an antifibrotic agent.
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