Efficient Heparin Recovery from Porcine Intestinal Mucosa Using Zeolite Imidazolate Framework-8

Heparin is one of the most valuable active pharmaceutical ingredients, and it is generally isolated from porcine intestinal mucosa. Traditionally, different types of commercial resins are employed as an adsorbent for heparin uptake; however, using new, less expensive adsorbents has attracted more interest in the past few years to enhance the heparin recovery. Zeolite imidazolate framework-8 (ZIF-8), as a metal–organic framework (MOF) with a high surface area, porosity, and good stability at high temperatures, was selected to examine the heparin recovery. In this research, we demonstrate that ZIF-8 can recover up to ~70% (37 mg g−1) of heparin from porcine intestinal mucosa. A mechanistic study through kinetic and thermodynamic models on the adsorption revealed appropriate surface conditions for the adsorption of heparin molecules. The effect of different variables such as pH and temperature on heparin adsorption was also studied to optimize the recovery. This study is the first to investigate the usage of MOFs for heparin uptake.


Introduction
Heparin (Scheme 1) is a linear, sulfur-rich polysaccharide in the glycosaminoglycan family with varying lengths and weights ranging from 2000-40,000 kDa; it is generally procured from the bovine and porcine intestinal mucosa. Heparin is predominantly used as an anticoagulant and antithrombotic agent to treat a variety of medical conditions, such as systemic embolism syndrome and deep vein thrombosis; however, it also has applications as an anti-inflammatory and antiviral drug. Furthermore, heparin has been demonstrated to inhibit cancer cell growth and delay the onset of the detrimental symptoms of Alzheimer's disease [1][2][3][4].
In this research, we report the recovery of heparin from the porcine intestinal mucosa using two types of ZIFs (ZIF-8 and ZIF-67) with different surface areas. Our results demonstrated that the ZIF with a higher surface area (ZIF-8) showed a higher amount of heparin recovery. We also evaluated the effects of several variables, such as pH and temperature, to investigate the most optimal conditions for heparin recovery. Our results further illustrated that the heparin recovery with ZIF-8 stabilized after five cycles. We also utilized a sheep plasma test to measure and compare the anticoagulant potency of ZIF-8 with commercial Amberlite FPA98 Cl, the results of which confirmed the significant potential of MOFs in the heparin uptake industry.

ZIF-8 and ZIF-67 Characterization
Scanning electron microscope (SEM) images of ZIF-8 and ZIF-67 were taken at the ACS Materials Facility using a Hitachi S-4800 scanning electron microscope (SEM) at 10 kV. The SEM images are shown in Figure 1. The infrared spectrum of ZIF-8 is shown in Figure 2.
In this research, we report the recovery of heparin from the porcine intestinal mucosa using two types of ZIFs (ZIF-8 and ZIF-67) with different surface areas. Our results demonstrated that the ZIF with a higher surface area (ZIF-8) showed a higher amount of heparin recovery. We also evaluated the effects of several variables, such as pH and temperature, to investigate the most optimal conditions for heparin recovery. Our results further illustrated that the heparin recovery with ZIF-8 stabilized after five cycles. We also utilized a sheep plasma test to measure and compare the anticoagulant potency of ZIF-8 with commercial Amberlite FPA98 Cl, the results of which confirmed the significant potential of MOFs in the heparin uptake industry.

ZIF-8 and ZIF-67 Characterization
Scanning electron microscope (SEM) images of ZIF-8 and ZIF-67 were taken at the ACS Materials Facility using a Hitachi S-4800 scanning electron microscope (SEM) at 10 kV. The SEM images are shown in Figure 1. The infrared spectrum of ZIF-8 is shown in Figure 2.

Heparin Adsorption Studies
Prior to optimization, it was important to evaluate the affinity of heparin toward each absorbent in a pure heparin solution. The results ( Figure 2a) demonstrated that ZIF-8 could adsorb more than 2-fold more heparin compared to ZIF-67 in a pure aqueous solution. The reported BET surface areas for ZIF-8 and ZIF-67 are >1300 and ~316 m 2 /g, respectively [31,32], which are well correlated with their differences in adsorption efficiency. Due to the superior adsorption capabilities of ZIF-8 over ZIF-67, ZIF-8 was selected for further experiments to evaluate adsorption in real samples containing porcine intestinal mucosa. The FTIR spectrum of ZIF-8 shows a band at 1712 cm −1 , which confirms the presence of C=N bond stretching. The bands at 1100 cm −1 and 990 cm −1 are attributed to C-N stretching. Additionally, a preabsorbed sample of ZIF-8 was dried and analyzed via FTIR spectroscopy so as to ensure that heparin adsorption occurred (Figure 2b). The resulting spectrum was compared with the FTIR spectrum of pure sodium heparin salt and the FTIR spectrum of ZIF-8 powder. The appearance of a band at 1712 cm −1 in the ZIF-8-heparin mixture is attributed to the C=O group of heparin, and the appearance of a band at 1222 cm −1 is attributed to the S=O group of heparin, which confirms the adsorption of heparin on the surface of ZIF-8.

Heparin Adsorption Studies
Prior to optimization, it was important to evaluate the affinity of heparin toward each absorbent in a pure heparin solution. The results ( Figure 2a) demonstrated that ZIF-8 could adsorb more than 2-fold more heparin compared to ZIF-67 in a pure aqueous solution. The reported BET surface areas for ZIF-8 and ZIF-67 are >1300 and~316 m 2 /g, respectively [31,32], which are well correlated with their differences in adsorption efficiency. Due to the superior adsorption capabilities of ZIF-8 over ZIF-67, ZIF-8 was selected for further experiments to evaluate adsorption in real samples containing porcine intestinal mucosa. The FTIR spectrum of ZIF-8 shows a band at 1712 cm −1 , which confirms the presence of C=N bond stretching. The bands at 1100 cm −1 and 990 cm −1 are attributed to C-N stretching. Additionally, a preabsorbed sample of ZIF-8 was dried and analyzed via FTIR spectroscopy so as to ensure that heparin adsorption occurred ( Figure 2b). The resulting spectrum was compared with the FTIR spectrum of pure sodium heparin salt and the FTIR spectrum of ZIF-8 powder. The appearance of a band at 1712 cm −1 in the ZIF-8-heparin mixture is attributed to the C=O group of heparin, and the appearance of a band at 1222 cm −1 is attributed to the S=O group of heparin, which confirms the adsorption of heparin on the surface of ZIF-8.

Effect of pH
One of the most important parameters to consider when studying any adsorption/ desorption system is pH, as pH fluctuations can have significant impacts on adsorption efficiency. For this reason, the effect of pH on the adsorption of heparin onto ZIF-8 was investigated by changing the pH of the real sample of the heparin solution from 3 to 11 in a series of experiments; adsorption efficiency and capacity were then evaluated. The results ( Figure 3a) indicate that heparin adsorption increases when increasing the pH from 3 to 8. The highest adsorption was observed at a pH of 8, where an efficiency of 57.8% and a capacity of 30.4 mg g −1 were recorded; efficiency and capacity subsequently remained constant as more alkaline conditions were induced. Notably, increasing pH slightly deprotonates heparin, resulting in more negative charges that make it more receptive to the positive active sites of ZIF-8. We selected pH = 8 as the optimal pH since the stability of MOFs subsequently decreases at higher pH conditions. sults ( Figure 3a) indicate that heparin adsorption increases when increasing the pH from 3 to 8. The highest adsorption was observed at a pH of 8, where an efficiency of 57.8% and a capacity of 30.4 mg.g −1 were recorded; efficiency and capacity subsequently remained constant as more alkaline conditions were induced. Notably, increasing pH slightly deprotonates heparin, resulting in more negative charges that make it more receptive to the positive active sites of ZIF-8. We selected pH = 8 as the optimal pH since the stability of MOFs subsequently decreases at higher pH conditions.

Effect of the Adsorption Dosage
ZIF-8 showed a remarkable capacity in the removal of contaminants. In this part of the study, we investigated the effect of the adsorbent dosage of ZIF-8 on heparin uptake by varying the amount of ZIF-8 from 100 to 1000 mg. The results (Figure 3b) indicated that increasing the adsorbent dosage to 500 mg caused the adsorption efficiency and capacity to increase to 58.2% and 30.6 mg.g −1 , respectively. Increasing the dosage of the adsorbent further led to a slight decrease in the capacity while efficiency remained constant. Notably, a higher adsorbent dosage increased the adsorption due to a higher surface area and the presence of more active sites on ZIF-8 adsorbents; the adsorption capacity, however,

Effect of the Adsorption Dosage
ZIF-8 showed a remarkable capacity in the removal of contaminants. In this part of the study, we investigated the effect of the adsorbent dosage of ZIF-8 on heparin uptake by varying the amount of ZIF-8 from 100 to 1000 mg. The results (Figure 3b) indicated that increasing the adsorbent dosage to 500 mg caused the adsorption efficiency and capacity to increase to 58.2% and 30.6 mg g −1 , respectively. Increasing the dosage of the adsorbent further led to a slight decrease in the capacity while efficiency remained constant. Notably, a higher adsorbent dosage increased the adsorption due to a higher surface area and the presence of more active sites on ZIF-8 adsorbents; the adsorption capacity, however, decreased at dosages higher than 500 mg. This phenomenon is attributed to the interference of other components in the sample, namely, dermatan and chondroitin sulfate, which compete with heparin and prevent it from being adsorbed onto the surface of ZIF-8. Furthermore, the aggregation of ZIF-8 at a higher dosage may also serve to decrease the active sites of the adsorbents for heparin molecules. In addition, the observed decrease in the adsorption capacity from 35 to 15 mg g −1 is the result of increasing the mass (m) in Equation (4) [17].

Adsorption Time and Temperature Effects
The results from the contact time experiment (Figure 3c) indicated that when increasing the contact time, the adsorption efficiency and capacity increased to 64.5% and 33.9 mg g −1 , respectively, after 300 min of contact time. This trend was anticipated due to the length of time that it takes heparin molecules to diffuse from the solution and bond to ZIF-8 adsorbent surfaces. As observed in the experiment, adsorption approached stability after that, revealing that ZIF-8 had reached its full capacity at these experimental conditions. In addition to contact time, the solution temperature is another highly influential parameter in adsorption systems. Therefore, to study its effect on heparin uptake, a series of experiments were performed by varying the temperature from 25 • C to 75 • C while keeping all other parameters constant. The results (Figure 3d) demonstrated that by increasing the temperature to 55 • C, the adsorption efficiency and capacity increased to 70% and 36.8 mg g −1 , respectively. By increasing the temperature, the heparin molecules can move more quickly in the solution due to a decrease in the solution's viscosity, which facilitates their interaction with the adsorbent surface. The minimal decrease observed after 65 • C is likely due to the decomposition of heparin molecules from temperatures that were too high to support its structural stability.

Kinetic and Thermodynamic Studies
In order to better comprehend the observed adsorption phenomena of ZIF-8 and its associated mechanisms, the collected data were analyzed via the following kinetic models: pseudo-first-order, pseudo-second-order, intraparticle diffusion, and Elovich (Equations (5)-(8); Figure 4 and Table 1). The obtained data, which are presented in Figure 4 and Table 1, show that the adsorption of heparin onto ZIF-8 best fits the pseudo-secondorder kinetic model since the R 2 value is much closer to 1.0 and q e(cal) is closest to q e(exp) out of all the examined models. Based upon this model, the rate-limiting step is the surface adsorption that involves chemisorption; therefore, the adsorption process is not limited by the concentration of heparin but rather the adsorption capacity of ZIF-8 [32]. This confirms the decrease in heparin uptake with increasing adsorption dosages ( Figure 3b). Further, the Elovich model also exhibits a good fit, supporting the adsorption of heparin on the ZIF-8 heterogeneous surface since this model assumes that the rate of the adsorption of the solute decreases exponentially as the amount of adsorption increases.
Molecules 2022, 27, x FOR PEER REVIEW 7 of 12 plotting lnKc versus 1/T and ln(1 − θ) versus 1/T [33,34]. These data are presented in Figure  5 and Table 2.   The positive values of ΔH and ΔS indicate the endothermic nature of the adsorption process and the high affinity of heparin molecules toward ZIF-8. The positive value of Ea also further confirms the endothermic nature of the adsorption process, while the negative values of ΔG reflect the feasibility of the process and its spontaneous nature [35].  The positive values of ∆H and ∆S indicate the endothermic nature of the adsorption process and the high affinity of heparin molecules toward ZIF-8. The positive value of E a also further confirms the endothermic nature of the adsorption process, while the negative values of ∆G reflect the feasibility of the process and its spontaneous nature [35].

Sorbent Reusability
ZIF-8 showed impressive performance in heparin uptake in real biological samples, with a~70% adsorption rate and a 37 mg g −1 capacity under optimized conditions. In order to evaluate the industrial potential of ZIF-8, we tested the stability and reusability of ZIF-8 through five adsorption/desorption cycles and harsh regeneration conditions. The results ( Figure 6) indicate that the ZIF-8 adsorbent has high stability and reusability even after five adsorption/desorption cycles and exposure to harsh regeneration conditions.

Sheep Plasma Clotting Assay
The porcine intestinal mucosa solution used for the sheep plasma clotting assay was prepared via a previously established and reported method [18]. The desired amount of adsorbent was mixed with the mucosa, followed by the stirring of the mixture at 55 °C for 3 h. Lastly, the adsorbent was filtered and washed with Milli-Q water. The solid heparin was then eluted from the adsorbent as described in a previous publication [18]. A similar method was employed for the commercial Amberlite resin. The anticoagulant potency of the recovered heparin from both ZIF-8 and the Amberlite resin was measured and compared [19]. The results indicated that the total activity of the eluted heparin utilizing the ZIF-8 adsorbent was 64 ± 1.8 U per gram of mucosa compared to 59 ± 2.6 U per gram of mucosa from the commercial Amberlite FPA98 Cl resin. These results highlight the promising potential of the ZIF-8 adsorbent in an industrial setting.

Materials
Heparin sodium salt of analytical grade was purchased from VWR, USA. Commercially synthesized ZIF-8 and ZIF-67 were purchased from ACS Material, LLC (Pasadena, CA, USA). Milli-Q water was used for the preparation of all solutions and the adsorption experiments. Porcine intestinal mucosa (heparin content of ~1300 mg L −1 ) was purchased Adsorption efficiency (%) Adsorption capacity (mg.g⁻¹) Figure 6. Stability test of heparin recovery over ZIF-8.

Sheep Plasma Clotting Assay
The porcine intestinal mucosa solution used for the sheep plasma clotting assay was prepared via a previously established and reported method [18]. The desired amount of adsorbent was mixed with the mucosa, followed by the stirring of the mixture at 55 • C for 3 h. Lastly, the adsorbent was filtered and washed with Milli-Q water. The solid heparin was then eluted from the adsorbent as described in a previous publication [18]. A similar method was employed for the commercial Amberlite resin. The anticoagulant potency of the recovered heparin from both ZIF-8 and the Amberlite resin was measured and compared [19]. The results indicated that the total activity of the eluted heparin utilizing the ZIF-8 adsorbent was 64 ± 1.8 U per gram of mucosa compared to 59 ± 2.6 U per gram of mucosa from the commercial Amberlite FPA98 Cl resin. These results highlight the promising potential of the ZIF-8 adsorbent in an industrial setting.

Materials
Heparin sodium salt of analytical grade was purchased from VWR, USA. Commercially synthesized ZIF-8 and ZIF-67 were purchased from ACS Material, LLC (Pasadena, CA, USA). Milli-Q water was used for the preparation of all solutions and the adsorption experiments. Porcine intestinal mucosa (heparin content of~1300 mg L −1 ) was purchased from a local market. The subtilisin enzyme was purchased from STERM Company, Newburyport, MA, USA. Commercial Amberlite FPA98 Cl resin was purchased from Dow Chemical (Midland, MI, USA), USA. Sheep plasma, hydrochloric acid (37%), calcium chloride, analytical grade methanol, sodium hydroxide, and denatured ethanol were purchased from VWR and used as received. Heparin ELISA kits were purchased from MyBiosource, San Diego, CA, USA.

Instrumentation
A Spectrostar Nano microplate reader (BMG LABTECH, Cary, NC, USA) was used to measure the heparin concentration in both pure and real samples. MOFs, heparin sodium salt, and MOF-heparin were characterized by a Shimadzu IR Affinity FTIR instrument (Shimadzu Scientific Instruments, Columbia, MD, USA).

Solutions and Methods
A heparin stock solution (1000 ppm) was prepared by adding the required amount of heparin salt to Milli-Q water and used for standard experiments. This solution was used to investigate the adsorption capacity of each adsorbent in the standard condition. For this, 0.5 g of ZIF-8 and ZIF-67 were added to separate solutions, the two mixtures were stirred for 5 h at 55 • C in an incubator, and the adsorbed heparin was calculated for both mixtures by using the methylene blue method according to the previously published procedure [18,19]. Specifically, to examine the material's absorption capabilities, 250 mg of the MOFs was added to a 25 mL aqueous solution of 1000 ppm heparin sodium. In the incubator, the solution was stirred for 3 h at 55 • C. Five milliliters of the solution was removed and filtered using a syringe filter, and the concentration of heparin in the supernatant was measured using the methylene-blue-assisted spectrophotometric method. Methylene blue (MB) is a cationic metachromatic dye that can bind to heparin. First, 1 mL of the heparin solution was added to 1 mL of MB solution (10 mg L −1 ). Then, the solution was mixed using a vortex mixer for 10 min, and the absorbance was measured by a plate reader based on the intensity of the band at λ max = 663 nm, which is attributed to the free MB. When MB forms a dimer with heparin, the intensity of the band at 663 nm decreases due to the lower concentration of free MB. The concentration of heparin after adsorption was calculated using the pre-plotted standard calibration curve.
The adsorption efficiency of heparin on the ZIFs and the anticoagulant potency of the eluted heparin were evaluated in porcine intestinal mucosa by using sheep plasma and heparin ELISA kits. The detailed adsorption process can be found in our previous publication [18].
The adsorption efficiency and capacity were calculated via Equations (3) and (4): Adsorption capacity q e = where C 0 and C e (mg L −1 ) are the initial and equilibrium heparin concentrations (measured with the ELISA kit), V (L) is the volume of the mucosa solution that was used for heparin adsorption, and m is the mass of the adsorbent used (g). The experimental conditions of the adsorption were optimized through different influential parameters, such as pH, contact time, and temperature. Furthermore, in order to understand the mechanism of adsorption and also the commercial viability of the adsorbent, other parameters such as adsorbent dosage, kinetic and thermodynamic properties, and the reusability of the absorbent material were investigated. All experiments were carried out in triplicate, and the error bars in the figures indicate the standard deviation of each experiment. Pseudo-first-order, pseudo-second-order, intraparticle diffusion, and Elovich models were calculated to have a better understanding of the adsorption phenomena. The linearized equation of each model is given in Equations (3)-(6), respectively. The pseudofirst-order model (Equation (5)) considers a proportional relation between the occupation of adsorption sites and the number of unoccupied sites.
log q e − q t = log q e − K 1 2.303 × t In Equation (5), the value of K 1 (min −1 ; the rate constant of the second-order equation) and q e can be obtained from the slope and intercept of the linear plot of log q e − q t versus t, respectively, where q e (mg g −1 ) and q t (mg g −1 ) are the adsorbed heparin at equilibrium and at any time t, respectively [36].
The pseudo-second-order kinetic model assumes that the adsorption rate is not dependent on the heparin adsorption but the adsorption capacity of ZIF-8.
In Equation (6), K 2 (g mg −1 min −1 ) and q e (mg g −1 ) are the rate constant of the second-order equation and the maximum adsorption capacity, respectively, which can be calculated from the slope and intercept of the plot of t/q t vs. t [37][38][39].
In the intraparticle diffusion model (Equation (7)), the limiting steps are considered to be mass transport.
In this model (Equation (8)), the values of K diff (the rate constant of intraparticle diffusion (mg g −1 min −1 )) and C (thickness of boundary) are calculated from the slope and intercept of the plot of q t versus t 1/2 [40]. The Elovich kinetic model (Equation (4)) considers the sorbent to have heterogeneous active sites with varying active energies during the adsorption process. q t = 1 β ln(αβ) + 1 β ln(t) (8) In this model (Equation (8)), the values of α and β (Elovich constants) can be calculated from the slope and intercept of the plot of q t versus ln(t) [41].
To evaluate the stability of the ZIF-8 adsorbent, after the first adsorption/desorption cycle, ZIF-8 adsorbent was regenerated by washing with a saturated NaCl solution for 3 h at 50 • C following the wash with Milli-Q water. The solution was tested with absorption spectroscopy to ensure that no heparin was carried to the next cycle. The same process was repeated across all five cycles [17].

Conclusions
Here, we report the use of ZIFs for heparin recovery, which is the first example of heparin recovery utilizing an MOF. To the best of our knowledge, these MOFs have been extensively used for various applications but not for heparin recovery. We employed two different MOFs with different surface areas, and the results met our expectations. ZIF-8, an MOF with a larger surface area than that of ZIF-67, proved to be more effective at adsorbing heparin, with approximately 70% adsorption capacity. Furthermore, our results revealed that the heparin recovered from ZIF-8 had higher total anticoagulant activity compared to the heparin recovered from the commercially available Amberlite FPA98 CI resin (sheep plasma clotting assay). The mechanistic adsorption study demonstrated that the adsorption process adheres to the pseudo-second-order kinetic model and is both thermodynamically endothermic and feasible. We also observed the effects of different variables, such as pH, temperature, and adsorbent dosage, on heparin recovery so as to determine the most optimal conditions for heparin uptake.
Author Contributions: M.K.A.: supervision, conceptualization, methodology, validation, investigation, data curation, visualization, writing-original draft, writing-review and editing, and funding acquisition. D.G.: supervision, writing-original draft, writing-review and editing, visualization, validation, investigation, and data curation. A.S.: writing-original draft, writing-review and editing, visualization, methodology, validation, investigation, and data curation. C.A.V.: methodology, validation, investigation, data curation, and writing-review and editing. D.P.K.: methodology, validation, investigation, and data curation. All authors have read and agreed to the published version of the manuscript.