Confirmation of Photo-Tailed Low-Molecular Weight Heparin

1. Introduction

Polysaccharides are diverse biopolymers characterised by a range of molar masses and corresponding viscosities. Due to their inherent flexibility and biocompatibility, they are used in many industries, including food, medicine, and materials science [1]. Today, ionising radiation is frequently used to modify the structure of polysaccharides, with molar mass playing a vital role in achieving specific results. Intrinsic viscosity is often measured to understand how polysaccharides are shaped in solution, as it gives information about their molecular weight, size, shape, and interactions [2].

Recent studies show that gamma and electron beam irradiation are eco-friendly methods for breaking down polysaccharides without the need for additives. These methods reduce the molecular weight, increase the polymer's solubility, and alter its biological activities, including antioxidant and hypolipidemic effects. Consequently, irradiation enables the tailoring of polysaccharides for food science and medicine, optimising their solubility or specific bioactivities as needed. [2,3,4].

Heparin, a highly sulphated polysaccharide, exists in several molecular weight ranges. Low-molecular-weight heparins, LMWH, are smaller molecules that primarily inhibit coagulation factor Xa rather than multiple clotting factors simultaneously [5,6]. Because of their reduced size, they work in a more controlled and predictable way. They cause less bleeding. These forms are safer and easier to utilise, making them particularly valuable in modern medical treatments [6,7].

Previous studies have shown that producing low-molecular-weight heparin via gamma or UVC irradiation offers advantages over traditional methods [8,9,10]. These radiation techniques use water, yield pure heparin fragments with lower molecular weight and toxicity, and are better for some medical uses [9,10]. Notably, Gamma or UVC irradiation creates heparin fragments with intermediate or low molar mass that are unsulfated or only slightly sulfated [10]. These fragments are mainly used to develop non-anticoagulant heparin derivatives for a range of therapeutic uses, including anti-inflammatory and anti-complement activities, tissue regeneration and wound healing, anticancer and anti-metastatic effects, biomedical device surface modification, and antioxidant functions. [5,10]

While gamma and UVC-irradiation methods have been used to produce low-molecular-weight heparin, the solution conformation of the resulting fragments has not been thoroughly examined. This study investigates the conformational behaviour of UVC-irradiated low-molecular-mass heparin in dilute solution. UVC-degradation is presented as a more controlled, efficient, and safer alternative to standard cobalt-60 gamma irradiation for tailoring LMWH.

Specifically, this study examines how low-molecular-weight heparin changes its shape and structure following photodegradation. Section 2 explains the experimental methods, including materials, viscosity measurements, molar mass analysis, UVC-irradiation, and conformational modelling. Section 3 shows the results, beginning with the kinetics of photodegradation and conformational stability. It also discusses how molecular weight affects modified heparin, explores scaling behaviour, and looks at chain flexibility and phototailing effects. The main findings are then discussed, and their significance is emphasised in Section 4.

2. Materials and Methods

2.1. Chemicals and Preparation of Solutions

Glentham Life Sciences Ltd. (UK) supplied commercial-grade heparin sodium salt powder. Surechem Products Ltd. (UK) provided sodium nitrate (NaNO3) for use as the supporting electrolyte.

A 0.1 M NaNO3 stock solution was prepared using double-distilled water. Heparin was then dissolved in this solvent to achieve a native solution concentration ($C$) of 0.005 g/cm³. All solutions were stirred continuously and thoroughly with a magnetic stirrer to ensure complete dissolution and uniformity.

2.2. UVC-Irradiation Procedure

Photodegradation was performed in a specially designed UVC reactor (Fig. 1). The heparin solutions were irradiated in ambient air at room temperature. To monitor the degradation kinetics, samples were collected for viscosity analysis immediately after irradiation and at set intervals thereafter (see, for example, Refs. [10]).

Figure 1. Schematic illustration of a laboratory photodegradation reactor.

Figure 1. Schematic illustration of a laboratory photodegradation reactor.

2.3. Viscosity and Molar Mass Determination

An Ostwald viscometer (YUCHENGTECH) with a 0.5 mm capillary and a solvent flow time of 100-150 seconds was used for the tests. A TCB-7 viscometer bath (PSL-Rheotek) was employed to maintain a constant temperature with ±0.01 °C. Flow times for both the solvent ($t_0$) and the solution ($t$) were measured three times to ensure reproducibility. Viscosity values were calculated as follows [11]:

$${\eta }_{\mathrm{r}}=t/t_0,{\eta }_{\mathrm{s}\mathrm{p}}={\eta }_{\mathrm{r}}-1$$

(1)

where ${\eta }_{\mathrm{r}}$ and ${\eta }_{\mathrm{s}\mathrm{p}}$ are recognised as the relative and specific viscosities, respectively.

The intrinsic viscosity ($\left[\eta \right]$) of both native and degraded solutions was determined via the single-concentration approach using the Solomon–Ciută equation [12]:

$$\left[\eta \right]=(2{\eta }_{\mathrm{s}\mathrm{p}}-{2\mathrm{l}\mathrm{n}\eta }_{\mathrm{r}}{)}^{0.5}/C$$

(2)

The relative weight-average molar mass ($M_{\mathrm{r}}$) was derived using the Mark-Houwink (M-H) equation [13,14]:

$$\left[\eta \right]$$

(3)

Using the literature values for the heparin- NaNO₃ system [15]: $K_{\left[\eta \right]}=$ 3.16 X ${10}^{-5}$ cm³/ g and $a=$ 0.88.

2.4. Photodegradation Kinetics

The overall first-order constant ($k_{\mathrm{s}}$) was derived from the slope of the linear regression of Eq. 4. The degradation lifetimes—half-life ($t_{1/2}$) and shelf-life ($t_{90}$) —were calculated using Equations 5 and 6, respectively [16,17]. Thermodynamic parameters, including the activation energy ($E_{\left[\mathsf{\eta }\right]}$), Arrhenius-like frequency factor ($A_{\left[\mathsf{\eta }\right]}$) and activation entropy (${\Delta S}^{‡}$), were determined through Equations (7) and (8) [18,19]:

Table 1. Kinetic Equations for Heparin Photodegradation.

Parameter	Equation	Eq. No.
Overall First-order Rate Law	$1$/$M_{\mathrm{t}}=1$/$M_0+k_{\mathrm{s}}{t}_{\mathrm{e}}$	(4)
Half-life	${t_{1/2}=\left[\eta \right]}_0/2k_{\mathrm{s}}=K_{\left[\eta \right]}{M}_{r,0}^a/2k_0$	(5)
$\mathrm{S}$helf-life (90%)	$t_{90}={0.1\left[\eta \right]}_0$/$k_{\mathrm{s}}={0.1K}_{\left[\eta \right]}{M}_{r,0}^a$/$k_{\mathrm{s}}$	(6)
Arrhenius-type relation	$\ln \left[\mathsf{\eta }\right]=\ln A_{\left[\mathsf{\eta }\right]}+$ $E_{\left[\mathsf{\eta }\right]}/\mathrm{R}T$	(7)
Eyring equation	$\ln A_{\left[\mathsf{\eta }\right]}=\ln {\displaystyle \frac{{\mathrm{e}\mathrm{k}}_{\mathrm{B}}T}{\mathrm{h}}}+{\displaystyle \frac{{\Delta S}^{‡}}{\mathrm{R}}}$	(8)

Table 1. Kinetic Equations for Heparin Photodegradation.

Parameter	Equation	Eq. No.
Overall First-order Rate Law	$1$/$M_{\mathrm{t}}=1$/$M_0+k_{\mathrm{s}}{t}_{\mathrm{e}}$	(4)
Half-life	${t_{1/2}=\left[\eta \right]}_0/2k_{\mathrm{s}}=K_{\left[\eta \right]}{M}_{r,0}^a/2k_0$	(5)
$\mathrm{S}$helf-life (90%)	$t_{90}={0.1\left[\eta \right]}_0$/$k_{\mathrm{s}}={0.1K}_{\left[\eta \right]}{M}_{r,0}^a$/$k_{\mathrm{s}}$	(6)
Arrhenius-type relation	$\ln \left[\mathsf{\eta }\right]=\ln A_{\left[\mathsf{\eta }\right]}+$ $E_{\left[\mathsf{\eta }\right]}/\mathrm{R}T$	(7)
Eyring equation	$\ln A_{\left[\mathsf{\eta }\right]}=\ln {\displaystyle \frac{{\mathrm{e}\mathrm{k}}_{\mathrm{B}}T}{\mathrm{h}}}+{\displaystyle \frac{{\Delta S}^{‡}}{\mathrm{R}}}$	(8)

2.5. Conformational Modelling

We evaluated chain conformations by using equations for the Self-Avoiding Walk Model (SAWM) and Random Walk Model (RWM), as shown in Table 2 [11,19,20,21]. The transition to theta conditions was described theoretically by Equation 18; This approach allowed determination of unperturbed dimensions and chain stiffness parameters, including the Flory characteristic ratio and persistence length, without adjusting the solvent [21].

3. Results and Discussion

3.1. Photo-Modification Kinetics

3.1.1. Degradation Rate and Lifetimes

Figure 2 depicts the photodegradation process inside the specified polymer-solvent system. The initial rapid decay phase is characterised by random chain scission events occurring at a roughly constant rate, signifying first-order kinetics [22,23]. According to the kinetic analysis presented in Figure 2, the rate constant ( $k_{\mathrm{s}}$) was calculated to be 583 min⁻¹ with a half-life ( $t_{1/2}$) of 4.29 min and a shelf-life duration ($t_{90}$) of 0.857 min [16,17].

3.1.2. Thermodynamic Interpretation

The Arrhenius-like plots in Figure 3 are used to compute the flow activation energy ($E_{\left[\mathsf{\eta }\right]}$), which is proportional to the slope of each line and describes how viscosity depends on temperature for different heparin fractions; the plot intercepts give the Arrhenius pre-exponential factors ($A_{\left[\mathsf{\eta }\right]}$), which are then evaluated with Eyring transition-state theory to calculate the activation entropy (${\Delta S}^{‡}$) [18,19,24]. The calculated values for activation energy, pre-exponential factor, and activation entropy for native (NM), small (SM), and ultra-small (USM) molar masses are listed in Table 3.

Figure 4. Arrhenius-like plots of three samples for aerobic UVC-degraded heparin sodium salt (0.005 g / cm3) in 0.1 M NaNO3 at 25°C; samples of native (NM, blue circles), small (SM, red circles), and ultra-small (USM, green circles) molar masses. The y-axis represents the natural logarithm of viscosity ($\mathrm{l}\mathrm{n}\left[\eta \right]$) and the x-axis is the inverse of temperature (1/T). The solid lines represent linear fits to the data for each molar mass group, with the corresponding regression equations displayed.

Figure 4. Arrhenius-like plots of three samples for aerobic UVC-degraded heparin sodium salt (0.005 g / cm3) in 0.1 M NaNO3 at 25°C; samples of native (NM, blue circles), small (SM, red circles), and ultra-small (USM, green circles) molar masses. The y-axis represents the natural logarithm of viscosity ($\mathrm{l}\mathrm{n}\left[\eta \right]$) and the x-axis is the inverse of temperature (1/T). The solid lines represent linear fits to the data for each molar mass group, with the corresponding regression equations displayed.

Table 3 shows that both the activation energy ($E_{\left[\mathsf{\eta }\right]}$) and the pre-exponential factor ($A_{\left[\mathsf{\eta }\right]}$) decrease simultaneously. As the molar mass drops from NM to USM, this points to a major change in how the system behaves. Specifically, the lower activation energy indicates the chains are more flexible and need less energy to flow [24]. Moreover, the less negative activation entropy (${\Delta S}^{‡}$) suggests the transition state is less restricted [19,24]. Taken together with lower$A_{\left[\mathsf{\eta }\right]}$ values, these results show that the heparin fragments become smaller and less complex as their molar mass decreases. From a biological perspective, this helps explain why low-molecular-weight heparins are more effective. Notify, the increased flexibility and simpler structures of USM and SM make them more suitable for advanced drug delivery systems that require rapid diffusion, sustained bioavailability, and stable in vivo performance.

3.2. Macromolecular Characteristics

3.2.1. Molecular-Weight Dependence and Size Behaviour

Figure 5 and Table 4 show that aerobic UVC irradiation gradually lowers both the radius of gyration and the hydrodynamic radius of heparin as its molar mass decreases. The NM, SM, and USM fractions also drop in a similar way, suggesting that the chain breaks are controlled and do not cause major changes in the shape, as indicated by the constant value of ${\rho }_{\mathrm{s}\mathrm{h}\mathrm{a}\mathrm{p}\mathrm{e}}$in Table 4. The stable structural ratio shows that the chain’s structure and flexibility remain intact after depolymerisation. These results suggest UVC irradiation can adjust molecular size while maintaining the structural stability, which is important for making functional low-molecular-weight heparin derivatives [25].

3.2.2. Impact on Critical Overlap and Thermodynamic Stability

Exposure to UVC light breaks down heparin chains, resulting in lower molar mass ($M$). As a result, the critical overlap concentration ($C^{*}$) increases, which improves the polymer's mixing with the solvent, as shown in Figure 6 and Figure 7 and Table 5 as well as in Ref [26]. Because the degraded chains (SM, USM) are smaller, they occupy less space in solution, requiring higher concentrations for overlap. Furthermore, higher second virial coefficients ($A_2$) indicate better solvation since the charged groups on these chains are more accessible [27].

3.2.3. Scaling laws and conformational regime

The logarithmic scaling connections between molar mass (M) and structural parameters depicted in Figure 8 provide significant insight into the conformational regime of photo-tailed heparin chains. The scaling plots show pronounced linearity. This linearity substantiates that the deteriorated samples exhibit power-law behaviour characteristic of flexible polyelectrolytes in dilute solution; see, for example, Ref. [3].

Table 6 shows the scaling exponents that describe the behaviour of the chains. A high radius-of-gyration exponent ($\nu =$ 1.30) means the chains expand a lot. This result suggests that UVC-treated heparin retains sufficient charge for electrostatic repulsion, preventing chain collapse, a behaviour characteristic of polyelectrolytes under screening conditions [28]. As established by de Gennes [29], the negative exponent for overlap concentration (${\nu }_{{\mathrm{C}}^{*}}=-0.86$ ) indicates that larger chains start to overlap at lower concentrations due to stronger excluded-volume effects. The second virial coefficient (${\nu }_{{\mathrm{A}}_2}=-0.12$ ) changes only a little with molar mass, which shows that 0.1 M NaNO3 is a good solvent. In summary, photo-modified heparin behaves like a charged polymer in a good solvent.

3.2.4. Ideal chain length parameters and chain flexibility

Figure 9 demonstrates how ideal chain parameters—end-to-end distance ($R_{\mathrm{e}\mathrm{ѳ}}$), characteristic ratio ($C_{\mathrm{\infty }}$), and persistence length ($l_{\mathrm{P}}$ )—relate to molar mass ($M$ ). Analysing these variables helps us understand how UVC irradiation changes the structure. As these values increase together, it suggests that longer chains become more extended and exhibit greater long-range stiffness [30,31,32].

Table 7 shows that native heparin molar mass (NM) has the highest persistence length, which means it is more rigid because of intramolecular electrostatic repulsion and a regular backbone structure [31,32]. The SM and USM fractions have lower persistence lengths, resulting in shorter, more flexible chains [3]. When the chain breaks, the polymer becomes less stiff, but its local structure remains ordered since only the long-range backbone is affected. Because of leftover charge effects, the polymer does not become a perfect random coil [31]. Still, the steady reduction in the persistence length indicates that it is becoming more flexible. In summary, these results show that UVC degradation mainly alters the size of heparin chains, rather than their local chemical structure [30]. Overall, these results indicate that UVC degradation primarily alters the size of heparin chains, rather than their chemical structure. The reduction in the unperturbed dimension$R_{\mathrm{e}\mathrm{ѳ}}$ , Flory ratio$C_{\mathrm{\infty }}$, and persistence length ($l_{\mathrm{P}}$) versus molar mass ($M$) shows that smaller fragments occupy less excluded volume and have lower local chain stiffness.

3.3. Conformational Consequences of Photo-Tailing

Scaling analysis and chain-length measurements show that photo-tailoring consistently changes the shape of heparin. When the molar mass decreases, the coils become smaller and more flexible, making the polymer more easily soluble in solvents and other molecules. The strong bond between the polymer and the solvent remains unchanged, so photochemical degradation does not appear to damage the groups needed for biological activity [32]. Photo-tailored low-molecular-weight heparin is more flexible and takes up more space, mainly due to electrostatic repulsion and excluded-volume effects [33].

3.4. Overall Structural Interpretation

In summary, the results show that UVC photo-tailing changes heparin’s shape in a manner consistent with predictions of polymer scaling theory [33]. As heparin moves from its natural state to very small molar masses, its chains get shorter, the overlap concentration increases, the interaction with the solvent gets better, and the chains become less stiff. These findings suggest that we can adjust heparin’s structure and physical properties through controlled photodegradation while retaining its polyelectrolyte properties [32].

4. Conclusions

This study shows that UVC photo-tailoring can alter the shape of heparin by lowering its molar mass, but its basic polyelectrolyte properties stay the same. Scaling analysis found that the broken-down fragments act like semi-flexible charged chains in a good solvent and follow typical power-law scaling. As molar mass went down, overlap concentration went up, and persistence length went down. This means the polymer chains became smaller and more flexible. Even after targeted photodegradation, the polymer stayed highly soluble in the solvent, which suggests better hydration and a stable shape.

In summary, controlled photo-tailoring is an effective way to change the structure of macromolecules. This method provides precise control over chain length, flexibility, and molecular behaviour in solution. Our results show that lowering the molecular weight with UVC irradiation changes the molecular shape. These findings offer a basis for developing low-molecular-weight heparin materials with improved physical and chemical properties for advanced medical and pharmaceutical uses.