Introduction

Conventional antiviral therapies which are available in the market are associated with numerous drawbacks. These chemical moieties pose adverse effects including gastrointestinal disturbances, altered hepatic and renal functions and variations in hematological parameters due to their repeated administration in higher doses in 24 h. Resultantly, patient compliance is compromised. In some instances, therapy is also discontinued. Therefore, modifying antiviral therapies can significantly impart advantages to these agents of miracle1. It’s a challenging task for pharmaceutical researchers to achieve desired dissolution rate of frequently administered drugs2. Traditional dosage forms are mostly linked with inconvenient dosing schedules and fluctuating drug levels in plasma. Through introduction of novel drug carriers, characteristics of drug moiety can be modified which helps in enhanced therapeutic outcomes in addition to patient adherence. This would ultimately result in less dosing frequency and controlled drug delivery at a predefined rate for an extended duration3. Properties of antiviral drug (Famciclovir) can be changed by loading in pH sensitive polymeric system to enhance its role as an antiviral therapeutic agent as the conventional drug dosage form seems to compromise the efficacy. Moreover, there was no research report of altering the dissolution rate of Famciclovir by loading in hydrogel (polymeric system). We have developed a unique pH-responsive polymeric network comprising Pluronic F127 for oral delivery of Famciclovir in a controlled manner.

Famciclovir is an antiviral drug being employed for treatment of Herpes Simplex and Varicella Zoster infections4. Prior to the discovery of Famciclovir, other antivirals were present in market which were administered to patients for more than three times a day. Famciclovir was developed to improve patient compliance as its dosing frequency was less, unlike previous drugs. However, its dosing frequency can further be reduced by encapsulating the drug in a hydrogel system. Famciclovir converts to Penciclovir inside the body5. In adults, its dose in active Herpes Zoster infection is 500 mg TID orally for 7 days. In case of genital Herpes Simplex virus, the drug is given three times per day orally at a dose of 250 mg for 7 to 10 days. Famciclovir is prescribed in BID at a dose of 250 mg for 7 days for genital chronic suppression therapy6. Famciclovir (FCV) is a nucleoside drug whose prescription enhanced dramatically during covid-19 onset. It was also prescribed in COVID-19 therapy as an add-on treatment7. The drug is soluble in water comprising various functional groups (amino, purine, and acetic acid groups)8. It is crystalline in nature. As the plasma elimination half-life of Famciclovir is 2–2.3 h (after oral administration) hence drug is given repeatedly, multiple times a day to maintain therapeutic drug levels in blood which adversely influences patient quality of life. Famciclovir is an inhibitor of growing DNA chain of virus. It competitively inhibits deoxyguanosine triphosphate for insertion in elongating viral DNA. Current therapies available in market are associated with drawbacks such as poor bioavailability (45–57%) and variable absorption. Maximum absorption of Famciclovir occurs in stomach as drug solubilizes at acidic pH9. Its short half-life, poor bioavailability results in variation of plasma drug concentration. These drawbacks compel the researchers to design better drug delivery systems for enhanced absorption and site specific distribution10.

Hydrogels can be defined in multiple ways. Monomers react together to synthesize hydrogels. These are combinations of polymers that are cross linked with each other and swell in aqueous media11. Hydrogels can also be defined as polymeric materials that has the capability to swell in water and can imbibe and retain a remarkable amount of water without themselves getting dissolved. Presence of high water contents inside hydrogels give them the remarkable property of flexibility and resemblance to natural tissues12. Hydrogels have been imparted with characteristics including hydrophilicity, ability to swell, permeability of molecules, and physical characteristics13. They are sensitive to their surrounding environment including temperature changes, magnetic and electric field variations, pH, concentration of biomolecules and light intensity thereby responsible for release of drugs which is loaded in them in a controlled manner14.

Hydrogels that are sensitive to their environment have huge potential in a variety of fields. pH and temperature are the environment stimuli that are found in human body. Hence, hydrogels that show response to specific pH and temperature can be easily deployed for controlled delivery of drugs at targeted areas15. Introduction of functional groups like amines, -COOH and imines in chemical structure of hydrogels can impart pH sensitivity in them. Physical and chemical properties of these groups are liable to change as they are capable of accepting or donating protons depending on pH of surrounding environment16.

Numerous synthetic polymers are utilized in the development of hydrogels owing to their non-toxic nature and biodegradability17. Pluronic F127 (PLU) comes from the family of block copolymers that are widely known as pluronics or poloxamers17. Pluronic F127 is soluble in water, white in color, odorless and tasteless. As it is a triblock copolymer so it contains polyethylene oxide-polypropylene oxide and polyethylene oxide18. Due to various properties of this block co-polymer, there seems to be increased interest in its utilization as drug delivery system. It is a nonionic surfactant and has hydrophilic groups in its structure. It can encapsulate a drug, alter its biodistribution, change the pharmacokinetics of the drug and also enhance drug transport through cellular pathways17. In addition to PLUF127, we have also used Methacrylic acid in our formulation. Methacrylic acid-based hydrogels are anionic materials showing pH responsiveness. Due to this, MAA is widely employed in controlled release drug delivery systems as it hinders degradation of drug at gastric pH inside stomach19.

Main objective of recent study was controlled delivery of Famciclovir by loading in a newly fabricated hydrogel system that can modify drug properties in a way which could enhance its therapeutic outcome. The short elimination half-life of Famciclovir is responsible for its increased dosing frequency which compromises patient compliance20. Its dosing frequency can be reduced if drug is loaded in polymeric network. Also controlled and site-specific drug delivery can be achieved with Famciclovir loaded pH sensitive hydrogels which is not possible in conventional dosage forms. Oral preparations currently present in market are linked with numerous side effects and drawbacks which decline its clinical effects. To the best of our information, no such research report has been presented until yet regarding successful loading and release of Famciclovir from hydrogel. Solid lipid nanoparticles loaded with Famciclovir were developed by Rawat et al. which showed in vitro release of drug up to 8 h only. They performed toxicity studies after 4 h of administration of their research product showing safe nature of solid lipid nanoparticles10. Gastroretentive drug delivery system for Famciclovir was also formulated by researchers that showed successful controlled drug release for 12 h21. In our work, controlled release of drug was seen for 36 h and that too at intestinal pH. In this way not only gastric side effects can be minimized but also less frequent administration can be achieved as more drug will be available at site of absorption. Additionally, in vivo toxicity study for different organs (after 14 days of administration of drug) was also executed confirming biosafety of newly formulated system.

Materials and methods

Materials

Famciclovir was gifted by ATCO Laboratories Limited, Pakistan. Pluronic F127 was provided by Sigma -Aldrich, Germany. Methacrylic acid (MAA) and Ammonium persulfate (APS) were bought from Merck, Germany. Potassium dihydrogen phosphate (KH2PO4) and N, N-Methylene bisacrylamide (MBA) were supplied by Sigma-Aldrich, UK. Hydrochloric acid (HCl) was taken from BDH Laboratory Supplies, England. Sodium hydroxide (NaOH), Potassium chloride (KCl) and Ethanol were sourced from Merck, Germany. All materials were of analytical grade. Freshly prepared distilled water was obtained from the Postgraduate Research Laboratory, University of Lahore.

Preparation of polymeric system

Hydrogels (polymeric system) were prepared by using free radical polymerization technique. Nine different formulations were prepared as mentioned in Table 1. The quantity of APS (initiator) was kept constant. Each formulation had a unique quantity of polymer (PLUF127), monomer (MAA), and crosslinker (MBA). Homogenous solutions of PLUF127, MBA and APS were prepared in distilled water. 5 ml water was used to make solution of PLUF127 and APS separately. While 10 ml water was added to beaker containing MBA. Beakers were placed on magnetic stirrers and stirring was done until clear solutions were made. Monomer was added to beaker containing polymer solution followed by addition of APS solution to start polymerization reaction. Finally, solution of MBA (crosslinker) was added with continuous stirring for another 10 min. Whole transparent solution was purged under nitrogen stream for 30 min to remove dissolved oxygen in a closed system. Then reaction mixture was transferred to glass test tubes which were placed in preheated water bath22. The temperature of the water bath was elevated sequentially to prevent bubble formation in a way that the reaction mixture was kept for 1 h at 55 ºC, for 6 h at 60 ºC and for 12 h at 65 °C23. After specified time period, test tubes were taken out of water bath, hydrogels were removed from test tubes and sliced into small discs of diameter 8 mm. Later, hydrogel discs were washed with ethanol and water mixture in the ratio of 1:1. Discs were placed in oven for drying at 55℃ for 48 h. Presumed structure of developed PLMA hydrogels is presented in Scheme 1.

Table 1 Concentration of polymer, monomer, initiator and crosslinker employed in development of PLMA hydrogels (1–9). Quantities mentioned in bold numbers mark the varying contents of respective formulation.
Full size table
Scheme 1
scheme 1

Presumed structure of PLMA hydrogel.

Full size image

Famciclovir loading into PLMA hydrogel

Famciclovir was loaded into hydrogels by adapting post drug loading technique. Phosphate buffer of pH 7.4 was chosen for preparation of 1% drug (Famciclovir) solution. Preweighed xerogel of each formulation was dipped in drug solution for 48 h at room temperature. After specified time interval, discs were taken out from drug solution, washed with distilled water and their weight recorded. Hydrogels were dried and their weight documented again24. % Drug loading was determined by following formula.

$$\% \, Drug \, loading = \frac{{\left( {Wf - Wi} \right)}}{Wi} \times 100$$
(1)

where, Wf is final weight after drug loading (dried discs), Wi expresses initial weight before drug loading.

Characterization of pluronic based polymeric system

Physical appearance of famciclovir loaded PLMA hydrogel and PLMA hydrogel without drug

For physical appearance, Famciclovir loaded and unloaded hydrogel was observed visually. Their texture, outer morphology and elasticity were examined with naked eye.

pH responsive swelling study

To ascertain the influence of pH on pluronic based hydrogels, swelling of PLMA hydrogels were conducted at 37 ºC in simulated gastric fluid of pH 1.2 and simulated intestinal fluid of pH 7.4. Preweighed hydrogel discs were placed in acidic and phosphate buffer of pH 1.2 and pH 7.4 respectively. After lapse of specific intervals i.e., 0.33, 0.5, 1, 1.5, 2, 2.5, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 24, 28, 32 and 36 h, hydrogels were removed from pH media, excess media was removed by using filter paper and weight was noted again. The degree of swelling, Qt at a time “t” was estimated by using the following equation25.

$${Q}_{t}= \frac{{m}_{t-}{m}_{o}}{{m}_{o}}$$
(2)

where mo tells the weight of dried hydrogels prior to placing in media i.e., at t = 0, mt denotes the weight taken after lapse of specified time t26.

Polymer volume fraction

Polymer volume fraction gives details about the polymer fraction which is in the complete swollen state. This is being denoted as v2, s. Equilibrium volume swelling was determined at pH 1.2 and pH 7.4 then following equation was applied to ascertain polymer volume fraction27;

$$v2,s = \frac{1}{Veq}$$
(3)

where, Veq represents equilibrium volume swelling.

Percent water content

Hydrogels have the capability to absorb large amount of fluid and swell to a significant degree. The absorbed fluid has influence on multiple parameters including drug loaded into the system. Thus, percent water content of hydrogel was estimated. Empty petri dishes and hydrogel discs in moist condition were weighed separately. Then hydrogels were placed in petri dishes and loaded petri dishes were put in an oven for drying at a specific temperature of 55℃. After 24 h, Petri dishes were taken out of the oven, lids placed on them and cooled in desiccator. Upon cooling, weight of hydrogel discs was noted again. Percent water content was determined by using this equation27;

$$w = {{\left[ {M_{cms} - M_{cds} } \right]} \mathord{\left/ {\vphantom {{\left[ {M_{cms} - M_{cds} } \right]} {\left[ {M_{cds} - M_{c} } \right] \times 100 = \left( {{{M_{w} } \mathord{\left/ {\vphantom {{M_{w} } {M_{s} }}} \right. \kern-0pt} {M_{s} }}} \right)}}} \right. \kern-0pt} {\left[ {M_{cds} - M_{c} } \right] \times 100 = \left( {{{M_{w} } \mathord{\left/ {\vphantom {{M_{w} } {M_{s} }}} \right. \kern-0pt} {M_{s} }}} \right)}} \times 100$$
(4)

where,

w = water content, percent.

Mcms = weight of petri dish and moist hydrogel.

Mcds = weight of petri dish and dried hydrogel.

Mc = weight of petri dish.

Mw = weight of water.

Ms = weight of hydrogel in dried state.

Yield %, gel% and gel time

Yield % and gel % were determined in order to document the quantity of reaction ingredients polymerized during the development of drug carrier. Preweighed dried discs (mi) from all formulations were placed in distilled water (100 ml) at 37℃ for seven days. Shaking was done occasionally. Later on, hydrogel discs were dried in a vacuum oven at a temperature of 40℃ until the achievement of constant weight. This helped in determination of water insoluble part. Then following formulae were applied28,29,30.

$${\text{Gel \% }} = { }\frac{{m_{d} }}{{m_{i} }} \times 100$$
(5)

where \({m}_{d}\) represents the dried weight of the formulated hydrogel disc after soaking for seven days, \({m}_{i}\) indicates the weight of the hydrogel disc before immersion.

$$Yield \% = \frac{Actual Yield}{{Theoretical Yield}} \times 100$$
(6)

where actual yield is the weight of the freshly prepared hydrogel, and theoretical yield is the total weight of reaction contents employed to develop a hydrogel.

Gel time indicates the duration of the period in which the reaction mixture started to transform into the hydrogel. Gel time helps in determination of progress of polymerization reaction. This time duration was noted for each formulation in minutes.

Famciclovir loading percent (%)

Famciclovir was loaded into polymeric system by employing post loading technique. One percent (1%) Famciclovir solution was prepared in phosphate buffer of pH 7.4 and preweighed hydrogel discs of all formulations were immersed inside it for 48 h at room temperature. After lapse of 48 h, discs were taken out form drug solution, washed with distilled water and weighed. Discs were dried in oven at 40 ºC for seven days24. The weight of loaded, dried hydrogel discs was again noted. Percent (%) drug loading was found by following formula:

$$\% \, Drug \, loading = \frac{{\left( {Wf - Wi} \right)}}{Wi} \times 100$$
(7)

Where, Wf is the final weight after drug loading, and Wi expresses the initial weight before drug loading.

Fourier transform infrared spectroscopy (FTIR) analysis

FTIR studies were carried out to investigate the interaction among Famciclovir, PLUF127, MAA and MBA by employing KBr pellet method (FTIR, Thermo Scientific Nicolet iN5, United States of America). These studies certified the occurrence of specific functional groups. These studies were performed for Famciclovir, PLUF127, MAA, MBA, Famciclovir loaded formulation and unloaded formulation31. Recording of spectra was done through the mode of transmittance in the wavenumber range of 4000–400 \({\text{cm}}^{-1}\)32,33.

Powder X-ray diffraction (PXRD) analysis

This technique determines the nature of sample whether it is crystalline or amorphous. The nature of the sample is responsible for water absorption by the hydrogel. If the result of the diffraction pattern is distinguished from those of independent components then it means the hydrogel has been designed properly34. PXRD analysis was carried out for PLUF127, Famciclovir loaded hydrogel and Famciclovir unloaded hydrogel. PXRD analysis was executed in a range of 10–80 2θ.

Scanning electron microscopy (SEM) analysis

SEM images can determine the surface characteristics of developed hydrogel. Hydrogels discs were scanned at different magnifications of 1.00KX, 2.01KX and 151X by employing SEM (instrument; JSm-6940, made in Tokyo, Japan)33.

Thermal analysis (TGA–DSC)

Thermal stability of hydrogel loaded with Famciclovir, hydrogel without loading of Famciclovir and individual Pluronic F127 were recorded by using TGA (Thermogravimetric analysis) and DSC (Differential scanning calorimetry). Thermal analyzer was used (Q600 TA V8.3 build101 Thermal Analysis System, TA Instruments, New Castle, DE, USA). Temperature range of 0-500ºC was provided at rate of 20℃ per minute35 and nitrogen was purged through the system at a rate of 20 ml/minute36.

In-vitro famciclovir release studies

Famciclovir release from Pluronic based polymeric system was estimated by deploying USP dissolution apparatus-II (Curio, Pakistan). For performing dissolution tests, simulated gastric fluid of pH 1.2 and simulated intestinal fluid of pH 7.4 were used. Dissolution was performed for 36 h for all formulations (PLMA-1 to PLMA-9). Famciclovir loaded hydrogels were placed inside dissolution vessels having 900 ml media at a temperature of 37 ± 0.5 ºC while the speed was adjusted at 50 rpm. At specific time intervals, samples (5 ml) from both fluids were drawn for detection of Famciclovir through UV visible spectrophotometer (Shimadzu, Germany) at \({\lambda }_{max}\) of 221 nm. Equal volume of fluid was replenished with same quantity for maintaining sink conditions. Comparison was generated between in vitro release profiles of hydrogel with that of conventional dosage form i.e., tablet (commercially available) to monitor the efficiency of formulations in comparison to the marketed tablet. All the data was obtained in triplicate. Following equation was applied to calculate Famciclovir release percentage37.

$$Famciclovir release \% = \frac{Absorbance of sample}{{Absorbance of s\tan dard drug solution}} \times 100$$
(8)

Acute in vivo toxicity studies

Guidelines given by the Organization of Economic Co-operation and Development (OECD) was strictly followed in order to conduct toxicological studies of developed polymeric networks. Approval of toxicity study protocol was taken from institutional research ethics committee (IREC) of Faculty of pharmacy, University of Lahore vide notification number IREC-2022-18. Six white, male (albino) rabbits were taken from the animal house of University of Lahore seven days before the conducting of studies in order to make them familiar to the environment of the laboratory. All the animals were kept at room temperature of 25 °C in addition to their exposure of light and dark period of 12 h. They were provided with adequate food and liquid (water). Animals were segregated into two groups i.e., the control group and the test group, with each group comprising of three members. Both the groups were kept on fasting for 12 h then the control group (group I) was only given food and water. On the other hand, the test group (group II) was administered finely ground PLMA hydrogel orally (5 g/kg) with water27. Animals were monitored for numerous physical parameters including weight, ocular toxicity, dermal irritation, illness, mortality, intake of water and food for a period of fourteen days36. These parameters are also presented in Table 4. After the mentioned time interval, samples of blood were drawn for examining biochemistry of blood including hematology, blood glucose, liver function test, renal function test and lipid profile. Later on, Animals were slaughtered and various vital organs including intestine, lungs, heart, right kidney, left kidney, liver, stomach, and spleen were preserved in 10 percent formalin solution for histopathological examination. Purpose of histopathological examination was to determine the biocompatibility of polymeric network (PLMA hydrogel) whether it damaged the cellular architecture of vital organs or is non-toxic to living tissues. Comparison was drawn between the results of the control group and the test group38.

Statistical analysis

Means ± standard deviation was applied in drug loading studies and in acute toxicity studies (biochemical analysis of blood). DD solver software was used to study drug release kinetics.

ARRIVE guidelines

The experimentation was conducted in accordance with applicable laws, and ARRIVE guidelines.

Results and discussion

Physical appearance of famciclovir loaded PLMA hydrogel and PLMA hydrogel Without drug

Freshly prepared PLMA hydrogels were of golden yellow color as shown in Fig. 1(A & B). Hydrogels were soft and elastic in nature. They possessed good integrity. Upon drying, their shape was retained. They did not undergo any cleavage even after absorption of fluid during swelling and dissolution studies. Hydrogels containing higher content of crosslinker were harder as compared to other formulations. Famciclovir-loaded hydrogels retained their shape after loading of drug as shown in Fig. 5.

Fig. 1
figure 1

PLMA Hydrogels of different formulations at dried state: (A) PLMA-1, (B) PLMA-4.

Full size image

pH responsive swelling study

In case of drug delivery systems, hydrogels showing higher degree of swelling are favored. The extent of swelling ratio of PLMA hydrogel determined the release behavior of drug in a controlled manner. The osmotic pressure gradient described the underline swelling phenomenon. pH-dependent swelling dynamics of fabricated hydrogels were determined in two types of medium i.e., acidic medium (pH 1.2) and phosphate buffer (pH 7.4) at 37℃. These studies were conducted to determine the response of polymeric system to external stimuli of change in pH. Figure 2 describes the physical appearance of hydrogel after they were placed in acidic and basic media. We noted swollen PLMA hydrogel at pH 7.4 while less swelling was observed at pH 1.2 as shown in Fig. 2. As all formulations comprised of varied ratios of PLUF127, MAA, and MBA, therefore, pH-dependent swelling behavior also showed a varied pattern from each other as depicted in Fig. 3 (A, B & C). Different swelling response was attributed to different pH as well as varied ratios of PLU F127, MAA, and MBA in nine hydrogel formulations. A higher degree of swelling was observed at a higher pH of 7.4 while minimal swelling was detected at pH 1.2. This was due to the carboxyl group in the structure of MAA. At acidic pH, the carboxyl group of MAA was supposed to be unionized. This resulted in a collapsed hydrogel system with no swelling at pH 1.2. At pH 7.4, the carboxyl group of MAA underwent ionization resulting in repulsion from each other, and hence swelling of the polymeric system was observed. This seemed to imply that if the pH of media was greater than the pKa of MAA, it would lead to ionization of MAA and ultimately increased swelling of hydrogel39. Such pH responsive swelling behavior of newly developed Pluronic based polymeric system might be useful in releasing drug slowly over an extended period of time.

Fig. 2
figure 2

Swelling study of PMLA hydrogel at pH 1.2 and pH 7.4.

Full size image
Fig. 3
figure 3

Comparative swelling behavior of PLMA hydrogels 1–9 formulations at pH 1.2 and at pH 7.4, (A) PLMA-1, PLMA-2, PLMA-3, (B) PLMA-4, PLMA-5, PLMA-6, (C) PLMA-7, PLMA-8, PLMA-9.

Full size image

Decrease in swelling ratio was observed when the content of Pluronic F127 was increased from 0.01 to 0.02 g and then 0.03 g (Fig. 3A). It must be noted that to assess the effect of different ratios of one content, the concentration of other reaction contents was kept the same. In this case of Pluronic F127 varied ratios in formulations PLMA-1, PLMA-2, and PLMA-3, the quantities of MAA and MBA were kept the same. Similarly, when ratios of MAA were varied in formulations having code PLMA-4, PLMA-5, and PLMA-6 then contents of MBA and PLUF127 were kept constant. Same practice was done in formulation codes of PLMA-7, PLMA-8, and PLMA-9, here MBA content was varied while keeping the ratios of PLUF127 and MAA constant. The decrease in swelling trend in formulations PLMA-1 to PLMA-3 where Pluronic F127 was increased from 0.01 to 0.03 g (MAA, MBA constant) might be due to the presence of polypropylene oxide which is hydrophobic and a subunit of PLUF127. This led to decline in pore size40. As the pore size was decreased so less intake of fluid was seen thereby minimizing the swelling index. Another reason for this behavior might be due to the formation of complex interpolymer masses41.

Ether group (in PLUF127) and carboxylic group (in MAA) showed potent interaction with each other leading to water absorption into the hydrogel. When PLUF127 concentration was increased it led to formation of complex structures with minimal aqueous channels. Hence a closed structure having increased content of PLUF127 (in hydrogel) was formed having complex polymer-monomer interactions with less number of channels restricting the entry of aqueous media into the hydrogel42,43.

The decline in swelling pattern was seen due to an elevation in MAA value from PLMA-4 to PLMA-6 (Fig. 3B). This might be due to the enhanced ratio of crosslinking of chains among the monomer itself and also among polymers with monomers. This high crosslinking density of polymeric network resulted in decline of flexibility of chains in the network. Thus, less fluid imbibition occurred in the system resulting in limited swelling of hydrogels. Maximum swelling was observed in formulation PLMA-4 having 5 g of MAA. With an increase in MAA content from PLMA-4 to PLMA-6 where the quantity of MAA was 5 g, 6 g and 7 g respectively, restricted swelling behavior was seen44.

From formulations PLMA-7 to PLMA-9 (Fig. 3C), ratio of MBA was altered keeping the ratios of other reaction contents the same. A rise in MBA ratio in PLMA-7 to PLMA-9 depicted decline in swelling pattern which was attributed to decreased porosity in the polymeric system. As crosslinking density was increased hence reduction in porosity hindered the entrance of swelling fluid into PLMA hydrogel leading to decline in swelling45.

Polymer volume fraction

High values of polymer volume fraction were obtained at pH 1.2 in contrary to pH 7.4 which might be due to swelling attitude. Fabricated polymeric system showed less swelling at pH 1.2 while a higher degree of swelling was observed at pH 7.4 so the polymer volume fraction was also high at pH 1.2. At pH 7.4, polymer volume fraction values were found to be low which indicated the significant swelling and profound expansion capacity of hydrogels. Polymer volume fraction increased with an increase in feed contents of PLUF127, MAA and MBA. Values were noted in triplicate and mean values are given in Table 2.

Table 2 Percent water content, percent drug loading, and polymer volume fraction (at pH 1.2 and pH 7.4) of all formulations.
Full size table

Percent water content

Hydrogels are supposed to retain some amount of water even in a completely dried state. Hence, water ratio determination is important to conduct experiments considering the real ratio factor. Percent water content decreased with an increase in ratios of polymer, monomer, and crosslinker as mentioned in Table 2. Incremental levels of PLUF127 led to the formation of complex structures thereby reducing the content of media in the system. Similarly, high levels of MAA and MBA resulted in hard structures with low water content. As with the change of the ratio of one content, other contents were kept the same so the percent water content values showed insignificant differences. Percent drug loading had a direct association with the percent water content in the system. Since our system is hydrophilic in nature, hence increase in water content enhanced the loading of drug. As soon as water content increased, maximum amount of solvent penetrated deeply into system which caused significant transfer of dissolved drug moieties inside the polymeric system so drug loading increased.

Yield%, gel % and gel time

Yield percentage, gel percentage, and gel time were estimated for all nine formulations to see the impact of varying concentrations of PLUF127, MAA and MBA on these parameters. To determine the influence of one variable the ratios of other variables were kept the same. Results have been shown in Fig. 4. In the formulation series (PLMA-1 to PLMA-3), enhancement in gel percentage was observed. Behavior of yield percentage was synonym to gel percentage. It also elevated with an increase in PLUF127 content in the hydrogel. This tells us the influence of polymer concentration on the density of cross-linking. As the mechanism adapted was free radical polymerization, PLUF127 behaved like macro radical and yield% and gel % were enhanced by increasing the ratio of PLUF127 which was credited to elevated ratio of reactant. However, gel time was found to decrease with an increase in concentration of PLUF127 as small quantity of MBA found it difficult to develop a cross-linking network among polymeric chains of PLUF127 and MAA as shown in Fig. 4A.

Fig. 4
figure 4

Effect of (A) pluronic F127, (B) MAA, (C) MBA, on gel %, yield %, and gel time.

Full size image

Figure 4B depicts the formulations from PLMA-4 to PLMA-6 where MAA concentration was enhanced. An increase in MAA content was found to have a direct relationship with yield% and gel% as both got elevated. Reason behind this phenomenon was presence of huge quantity of functionally active radicals on methacrylic acid. However, an inverse relationship was observed in case of gel time as gel time reduced with an increase in monomer concentration. This is related to the fact that pace of polymerization reaction was increased and more inter linking of polymeric chains was there at earlier stages46.

An increase in MBA content was made from formulation codes PLMA-7 to PLMA-9 and result was elevation in gel% and yield% (Fig. 4C). Higher ratios of MBA were responsible for increase in density of inter linking chains hence a dense mass was obtained. More molecules of MBA were available to take part in polymerization reaction as cross-linker agents thus giving higher yield% and gel%. On the contrary, gel time showed opposite behavior. Gel time was reduced with the enhancement of MBA ratio. As the polymerization (reaction) rate was increased due to more quantity of MBA, hydrogel synthesis (product) rate also got faster leading to reduction in gel time. Polymeric chains started interlinking with each other at a higher rate in the presence of increased ratios of MBA47.

Famciclovir loading percent (%)

Physical appearance of Famciclovir loaded PLMA hydrogel in dried state is shown in Fig. 5. Figure 6 presents the picture of Famciclovir loading into hydrogel matrices and its relation with varied contents of PLU F127, MAA, and MBA. As nine different formulations were prepared so amount of drug loaded ranged between 0.32 and 0.39 g. Loaded hydrogel amount ranged from 0.83 to 0.93 g depending upon the formulation. These loaded hydrogels were further employed for in vitro drug release studies and other characterization techniques. Maximum drug loading of amount 0.39 g was observed in PLMA-4. It must be noted that swelling was found directly proportional to drug loading. Formulation showing highest degree of swelling showed maximal drug loading and vice versa. Also, increase in water content enhanced the loading of drug. With enhancement in water content, increased volume of fluid entered into system thereby increasing drug loading. There is inverse relationship between polymer volume fraction and drug loading. Drug loading was decreased with elevation in polymer volume.

Fig. 5
figure 5

Famciclovir loaded PMLA-4 hydrogel in dried state.

Full size image
Fig. 6
figure 6

Influence on famciclovir loading percentage due to varied contents of (A) pluronic F127, (B) MAA and (C) MBA.

Full size image

Incremental elevation in Pluronic F127 content from PLMA-1 to PLMA-3 resulted in a decline in drug loading (71.31% to 62.65%). Rise in the ratio of MAA from PLMA-4 to PLMA-6 revealed a decrease in drug loading percentage (73.74% to 63.47%) as depicted in Fig. 6B. Decrease in Famciclovir loading because of increased ratios of Pluronic F127 and MAA may be due to formation of complex and dense structures having more interaction between polymer and monomer and among monomer–monomer. Such sort of complex masses resulted in reduction in aqueous channels thereby hindering imbibition of surrounding fluid. So, less drug loading was seen. Governing force in drug loading was swelling which was more when concentration of polymer was less. More drug will entangle in polymeric chains in presence of less quantities of PLUF127.

Enhancement in proportion of MBA from PLMA-7 to PLMA-9 led to decline in Famciclovir loading percentage (68.52% to 60.89%) as shown in Fig. 6C. This seemed to own the fact that higher ratio of MBA was responsible for elevation in density of crosslinking. As thick dense masses of cross-linked polymeric chains developed owing to more presence of MBA, this led to reduction in availability of free voids among polymeric chains. Compact, non-flexible structure was unable to imbibe a large quantity of fluid. As less volume intake happened so was the swelling, hence less drug loading was achieved36,48. PLMA-4 was considered to be an optimized formulation having maximal loading of drug up to 73.74% due to its highest swelling tendency. Conclusively, this polymeric nexus is considered to be a promising carrier for Famciclovir owing to its considerable encapsulation of the drug.

Fourier transform infrared spectroscopy (FTIR) analysis

Purpose of FTIR spectroscopy was to establish the possible interaction of Famciclovir with the polymer and monomer of hydrogel system. Successful encapsulation of Famciclovir into PLMA hydrogel was confirmed through FTIR spectra due to presence of characteristic functional groups. Spectra of Famciclovir, drug-loaded PLMA hydrogel and unloaded PLMA hydrogel, pure PLU F127, MAA and MBA were recorded as shown in Fig. 7. FTIR graph of pure PLUF127 had shown the presence of OH group, CH stretching, CO bond, COC and CH2 bending at different frequencies. A broad peak was observed at 3402 \({\text{cm}}^{-1}\) which was suggestive of the presence of the OH group. Absorption band was seen in the region of 2829–3036 \({\text{cm}}^{-1}\) which recommended the occurrence of CH stretching band here. Peak at 1653 \({\text{cm}}^{-1}\) indicated the presence of CO stretch. COC stretching vibrations were observed at 1163 \({\text{cm}}^{-1}\). Peak at 988 \({\text{cm}}^{-1}\) depicted the presence of CH2 bending.

Fig. 7
figure 7

FTIR spectra of (A) pluronic F127, (B) MBA, (C) MAA, (D) famciclovir, (E) PLMA hydrogel without drug, and (F) famciclovir loaded PLMA hydrogel.

Full size image

In case of methacrylic acid spectra, peak was observed at 2930 \({\text{cm}}^{-1}\) which was correlated with the occurrence of methyl CH stretching. Peak observed at 1632 \({\text{cm}}^{-1}\) was attributed to the presence of C = C stretching vibrations. Peak at 1425 \({\text{cm}}^{-1}\) was due to OH bend49.

FTIR spectra of MBA has been shown in Fig. 7B, N–H stretching vibrations can be seen at 3294 \({\text{cm}}^{-1}\) where a broad band is visible. A particular peak was observed at 1655 \({\text{cm}}^{-1}\) which recommended the presence of (–C = C–) group here. This lied within the range of 1620 \({\text{cm}}^{-1}\) to 1680 \({\text{cm}}^{-1}\). Peak at 2955 \({\text{cm}}^{-1}\) depicted –CH2 symmetric vibrations. Bands seen at 1539 \({\text{cm}}^{-1}\) and 1622 \({\text{cm}}^{-1}\) were affiliated with stretching of N–H and C = O groups of acrylamide respectively50.

FTIR spectra of Famciclovir showed specific peaks of characteristic wavenumbers. Peak at 3331 \({\text{cm}}^{-1}\) was reflection of O–H stretching. Peak at 2363 \({\text{cm}}^{-1}\) indicated the presence of O = C = O group stretching. Stretching band at 1726 \({\text{cm}}^{-1}\) confirmed the presence of C = O bond. At 1213 \({\text{cm}}^{-1}\) region, C-O stretching was visible. C = C bending was confirmed at peak of 957 \({\text{cm}}^{-1}\) area9.

Successful grafting of the polymeric network had been done through free radical polymerization as shown in FTIR spectra of unloaded PLMA hydrogel (Fig. 7E). Peaks were observed at 1701 \({\text{cm}}^{-1}\), 1244 \({\text{cm}}^{-1}\), 1175 \({\text{cm}}^{-1}\) regions depicting COOH, CH2-CH2 bend and C–O–C stretching vibrations respectively. A specific band that appeared at 1701 \({\text{cm}}^{-1}\) owed its existence to the carboxylic group of methacrylic acid. Presence of C–O–C, CH2-CH2 groups in fabricated Pluronic based hydrogel in region from 1100 to 1300 \({\text{cm}}^{-1}\) were attributed to successful grafting of Pluronic F127 into the system51. Various absorption bands ascertained the occurrence of C–O–C, C = O and CH2-CH2 groups from PLUF127 in the new polymeric system52. The region at 3431 \({\text{cm}}^{-1}\) and 3290 \({\text{cm}}^{-1}\) in PLMA hydrogel spectrum showed the presence of O–H and NH stretching vibrations respectively. O–H of PLUF127 at 3402 \({\text{cm}}^{-1}\) had shifted to a new position of 3431 \({\text{cm}}^{-1}\) in PLMA hydrogel. Similarly, N–H stretch of MBA at 3294 \({\text{cm}}^{-1}\) seemed to appear at 3290 \({\text{cm}}^{-1}\) in fabricated hydrogel. Development of new peaks and slight shifting of peaks demonstrated the successful fabrication of a new hydrogel. The loaded polymeric system also demonstrated the existence of Famciclovir by showing peaks at 1711 \({\text{cm}}^{-1}\), 957 \({\text{cm}}^{-1}\) which suggested the presence of C = O stretching vibration and C = C bending respectively (Fig. 7F). The specific peak of 957 \({\text{cm}}^{-1}\) of Famciclovir was present intact in PLMA hydrogel loaded with Famciclovir. Also, new peak developed in area of 3448 \({\text{cm}}^{-1}\) revealed the overlapping of O–H groups of Famciclovir and PLUF127. These results confirmed the existence of Pluronic F127 and entrapment of Famciclovir in hydrogel (PLMA) thus suggesting that all components of fabricated hydrogels were compatible with each other. Slight shifting of peaks was indicator of Famciclovir entrapment in PLMA hydrogel. Free radical polymerization might be a reason for peak shifting. This indicated that drug is compatible with the employed polymer53. Moreover, successful crosslinking of different compounds had occurred thereby developing a stable hydrogel system. Also, drug had been loaded into the system. Successful fabrication and loading of hydrogel confirmed the role of system as drug delivery carrier54.

Scanning electron microscopy (SEM) analysis

Scanning electron microscopy analysis was conducted to get information about the surface characteristics and microstructure of fabricated hydrogel. Images were recorded through SEM at numerous magnifications (Fig. 8A at 1.00KX, Fig. 8B at 2.01KX, Fig. 8C at 151X magnification). SEM images revealed rough, porous and irregular surface of hydrogel. Also, cracks were present on polymeric network. Presence of cracks helped in absorption of fluid from surrounding environment26. This imparted significant swelling behavior to formulated hydrogels. Due to absorption of fluid, drug was easily loaded into the system. This signifies that developed hydrogel can act as an excellent drug delivery carrier.

Fig. 8
figure 8

SEM images of PLMA hydrogel at (A) 1.00 KX magnification, (B) 2.01 KX magnification, (C) 151X magnification.

Full size image

Powder X-ray diffraction (PXRD) analysis

As shown in Fig. 9, PXRD results were obtained to get information about the crystallinity or amorphous nature of Pluronic F127, loaded PLMA, and optimized unloaded hydrogel matrix. The graph of PLUF127 demonstrated significant peaks at 18° and 23° which signified that the polymer was crystalline (Fig. 9A). No distinctive peaks were observed in loaded and unloaded hydrogel matrices as shown in graph (Fig. 9B, C). This certified the amorphous nature of fabricated loaded and unloaded hydrogel. Graph is in a diffused manner without intense signals revealing amorphous nature of developed polymeric nexus both in loaded and unloaded forms. Significant peaks of Pluronic F127 did not appear in fabricated nexus proving strong molecular interaction among the constituents and development of a polymeric complex. The obtained diffractogram proved successful integration of Famciclovir in the formulated nexus. Also, the crystalline state of Famciclovir was seen diminished owing to the development of new molecular interactions (which resulted due to free radical polymerization)52,55,56. Moreover, decline in crystallinity improved the swelling kinetics of loaded polymeric combinations. Hence, more imbibition of water was seen in developed nexus. The same results were also reported in other studies where crystallinity was reduced in hydrogel thus imparting amorphous characteristics to it57,58. Additionally, transition of Famciclovir from crystalline to amorphous phase resulted in modification of solubility of system hence solvent molecules found more chance to interact with polymeric network. Surface area of system was enhanced in amorphous state. This increased hydration of system leading to more drug loading and drug release. Dissolution rate and oral bioavailability increases in the presence of amorphous form of system59.

Fig. 9
figure 9

PXRD of (A) pluronic F127, (B) famciclovir loaded PLMA hydrogel and (C) unloaded PLMA hydrogel.

Full size image

Thermal analysis (TGA–DSC)

TGA was employed to determine the decomposition response of samples after the provision of heat (Fig. 10). Thermograms of pure PLUF127, Famciclovir-loaded PLMA hydrogel and unloaded PLMA hydrogel were obtained by providing them heat in a temperature zone of 0 to 500℃. Pluronic F127 showed a stable response till a temperature of 150℃ then it started decomposing. The decomposition rate got higher at 200 ℃ and onwards. While the decomposition rate was even more prominent and notable from 300 to 425℃. Sudden decomposition was revealed at higher temperatures. Multiple researchers have found decomposition response at 300 ℃ and onwards60. When TGA of PLMA hydrogel was exhibited, the thermogram showed loss of weight at initial stages at a slower pace which was attributed to water loss in the polymeric system. However, the polymeric nexus was much more stable than individual PLUF127 over an elevated temperature zone which signifies the successful fabrication of a stable polymeric system. Reduction in weight percentage was seen in four different phases. Loss of water from hydrogel resulted in loss of weight in primary phase where only 10 to 15% of weight loss was visible at 200℃. In phase II, 5% of weight reduction occurred at 225℃ owing to the presence of MAA in the hydrogel, then weight remained constant till 400℃. This picture is contrary to individual PLUF127 where significant decomposition happened at 300℃. Less than 35% of weight loss was observed till 400℃. Further weight reduction happened at 425℃ and onwards encompassing phases III and IV thus proving an elevation in the decomposition half-life of PLMA hydrogel in comparison to individual reactants. The existence of PEO-PPO-PEO block copolymer in PLUF F127 was responsible for the weight reduction of PLMA polymeric nexus. Fabricated hydrogel was found to be thermally stable owing to the successful integration of reactants in the form of a cross-linked polymeric combination. TGA of hydrogel loaded with Famciclovir was also executed and enhanced thermal stability of Famciclovir-loaded PLMA hydrogel was observed as compared to reactants. Decomposition temperature of Famciclovir is 205℃ but this temperature was enhanced in Famciclovir loaded hydrogel which indicated that MAA grafting to PLUF127 modified the decomposition temperature of Famciclovir61.

Fig. 10
figure 10

(A) DSC of pure pluronic F127 (B) TGA of pure pluronic, (C) DSC-TGA of PLMA hydrogel (D) DSC-TGA of famciclovir loaded PLMA hydrogel.

Full size image

In case of DSC, the endothermic peak was observed in the thermogram of PLUF127 at 65℃ which might be due to the breakage of the crystalline structure of the Pluronic F127 backbone (Fig. 10A). The wider endothermic peak at 310–375℃ demonstrated the degradation of the polymeric chain. Endothermic peak and exothermic peak were observed at 250℃ and at 420℃ respectively in the unloaded hydrogel. A peak at 250℃ in unloaded hydrogel depicted the decomposition of the carboxylic group of MAA monomer. DSC thermogram of Famciclovir loaded hydrogel disc was also exhibited which depicted the endothermic peak at 425℃. Thermograms of loaded and unloaded hydrogel discs revealed the shifting of peaks toward elevated temperatures (Fig. 10B, C). This recommends that developed hydrogel is more thermostable as it can tolerate high temperatures with less rate of decomposition as compared to individual reaction contents. During thermal analysis hydrogels were exposed to temperatures higher than 400 ℃ but such high temperatures aren’t seen in normal atmospheric conditions so we can claim that during storage at room temperature they will not degrade and retain their integrity.

In vitro famciclovir release studies

Famciclovir release from PLMA hydrogel was observed at pH of 1.2 (simulated gastric fluid) and pH of 7.4 (simulated intestinal fluid) for 36 h. As the physiological pH varies from acidic to neutral and then alkaline when we move from gastric to intestinal environment so same pH media were used for Famciclovir release studies. Drug release from the system was in minor quantities in the acidic buffer for all formulations for 36 h. In phosphate buffer, Famciclovir release was seen in a controlled way for a prolonged period as shown in Fig. 11. This seems to imply that developed hydrogel showed a pH-dependent response62.

Fig. 11
figure 11

In vitro Famciclovir release percentage at pH 1.2 and pH 7.4 from newly developed hydrogel (A) PLMA-1, PLMA-2, PLMA-3, (B) PLMA-4, PLMA-5, PLMA-6, (C) PLMA-7, PLMA-8, PLMA-9.

Full size image

In current study, drug release was seen for up to 36 h in the simulated intestinal fluid of pH 7.4 (Fig. 11). Controlled delivery of an antiviral drug (Famciclovir) can be achieved at elevated pH as the developed system has shown significant release of Famciclovir for a prolonged period (more than 90% in approximately all formulations for a period of 36 h). Percent Famciclovir release from all nine formulations in 36 h at pH 7.4 was found as PLMA-1 (98.56%), PLMA-2 (96.56%), PLMA-3 (94.41%), PLMA-4 (99.13%), PLMA-5 (96.56%), PLMA-6 (95.84%), PLMA-7 (96.56%), PLMA-8 (92.27%), PLMA-9 (90.12%) (Table 3).

Table 3 Percent drug release at pH 1.2, pH 7.4 and percent drug loading of PLMA hydrogel.
Full size table

The influence of different ratios of all contents of the hydrogel system on drug release was also evaluated. It was found that with an increase in PLU F127 content from PLMA-1 to PLMA-3, decrease in drug release was observed (Fig. 11A). Release of drug from the polymer system depends on its swelling behavior. The ratio of polymer present within the system governs the release of drug. Hydrogel matrices were eroded slowly thus releasing the drug for a prolonged period of up to 36 h in a controlled manner63. Increase ratio of polymer imparts the effect of prolonging the rate of release of drugs from the system which also revealed a controlled pattern. Hydrogel required more time to erode leading to its controlled behavior. A higher ratio of cross-linking occurred between the polymeric chains leaving behind less volume for drug diffusion and low penetration of surrounding fluid into the polymeric network64. Elevating the level of MAA from formulation PLMA-4 to PLMA-6 demonstrated less release of drug (Fig. 11B). This behavior was documented owing to a less swelling ratio pattern and decreased amount of drug encapsulated. Increased concentration of monomer resulted in the formation of a complex polymeric network having elevated levels of crosslinking density. As a result, less penetration of surrounding media is there hindering the escape of encapsulated drugs. our results are being supported by earlier studies too65. An increase in the MBA ratio led to a decrease drug release from matrices as we moved from PLMA-7 to PLMA-9 (Fig. 11C). The dense mass of crosslinking resulted in less swelling and ultimately lower drug release. Due to the increased ratio of MBA, the extent of cross-linking is elevated and surrounding media finds it difficult to permeate36. These results support the newly fabricated polymeric system for its use as a drug delivery carrier (as it can deliver the drug to targeted areas in a controlled way). Also, successful loading and release of Famciclovir in simulated intestinal fluid was confirmed through this study for all nine formulations declaring Famciclovir loaded PLMA hydrogel as a promising novel controlled drug delivery system which can be employed in treatment of Herpes Simplex and Varicella Zoster infections. As constant release of drug was found for period of 36 h hence, we can also say that this dosage form can help in reducing dosing frequency of Famciclovir.

Conventional dosage form (tablet) showed drug release behavior inferior to hydrogel system (Fig. 12). 98.71% of drug release was found in acidic pH of 1.2 while 96.70% of drug release occurred at pH 7.4 after lapse of 36 min. Drug release from marketed tablet was rapid and maximum amount got released in less than an hour (Table S3). However, in PLMA-4 (optimized formulation), 11.7 percent release occurred after 5 h in acidic buffer and 20.91 percent in pH 7.4 (Table S2). Hence drug release from newly created polymeric system was steadier, controlled and more constant in response to change in pH. Also, PLMA hydrogel showed drug release for 36 h but in conventional dosage form 100 percent drug release was attained in just 56 min.

Fig. 12
figure 12

In vitro Famciclovir release percent from marketed tablet at pH 1.2 and pH 7.4.

Full size image

Also, there is no possibility of burst release or lag phases because newly fabricated system has mesh like structure, Pluronic F127 and methacrylic caid has been crosslinked with each other by using MBA as cross linker. It’s a polymeric network/hydrogel where all components are entangled hence no chance of burst release. Additionally, alteration in quantities of monomer, polymer and crosslinker can be made for further optimization of system for more controlled and predictable release kinetics.

It must be noted that we have developed nine different formulations consisting of same ingredients but in varying quantities. Degree of swelling, percent drug loading and drug release data varied from each other owing to different quantities of ingredients employed and different pH of media utilized but overall behavior of drug swelling and release was same i.e., all formulations exhibited higher degree of swelling and drug release at simulated intestinal fluid (pH 7.4) and less swelling and release at simulated gastric fluid (pH 1.2). Also, all formulations followed zero order drug release (Table S1 & Supplementary Fig. 1). As our system didn’t swell in significant ratio at pH 1.2 so we expect that this system is not going to release sufficient drug in stomach rather it will release drug in small intestine so gastric side effects will be avoided as well as targeted drug delivery at the site of absorption will be achieved. Significant differences in drug release profiles were observed at pH of 1.2 and 7.4. But in case of similar pH media, all formulations exhibited release profiles with minimal differences.

Acute in vivo toxicity studies

Rabbits were selected as animal models to conduct acute toxicity studies. This study helped in the appraisal of the safety profile of the newly fabricated system. As the results of FTIR and thermal analysis confirmed polymer stability and lack of decomposition at physiological temperatures respectively so there were minimal chances of any immunogenic components or leachable toxins in the hydrogel. Hence hydrogels were assumed biocompatible which were later proved by acute toxicity study as well. During the entire study period of fourteen days, animals were being provided with controlled environmental conditions. Numerous parameters including the weight of the animal, dietary intake, fluid consumption, change in their behavior, and other health-related parameters such as dermal, and ocular irritation were strictly recorded.

Both the groups (including treated and control) remained healthy and stable during the study duration. No rabbit showed any sign or symptom of sickness. Minimal variations were observed in the case of dietary intake, and body weight, among the test and control groups as shown in Table 4. Both groups demonstrated normal behavior. Also, eyes and skin hypersensitivity were not seen. No mortality was there and signs of morbidity were also absent. The hematological report presented a normal count of all blood cells. Liver function tests, lipid profile, and renal profile presented a picture of the healthy function of these organs in both groups (Table 5). The results of both groups were comparable and lied within normal ranges of physiological values. This certifies that the developed drug delivery system is harmless to living tissues and biocompatible66. Histopathological studies were also conducted by developing slides of various vital organs of both groups and the results were promising. This served the purpose for determination of biosafety of newly developed polymeric system. No significant deviations in cell structure of all organs i.e., heart, liver, kidney, lung, spleen, small intestine was recorded in the hydrogel-administered group. All organs presented a normal picture as shown in Fig. 13. Cardiomyocytes had no apparent inflammation. No edema was observed in heart tissues. Cardiac structure was intact with no cellular necrosis. No mineralization was identified in pulmonary artery. Normal lung parenchyma bound by pleura and interlobular septa was seen. No edema was observed in bronchioles. Normal architecture of alveoli was found to possess thickened walls. Renal tissue degradation was absent. Tubules, ducts, and nephrons documented normal structure. The vasculature ratio was uniform. No evidence of inflammatory disease, granuloma or malignancy was seen. In the stomach, no ulceration was observed. Mucous secreting cells, and parietal cells showed structural integrity. Fundic glands were well organized. Pyloric glands maintained their structure with no inflammation. Hepatocytes were present in an organized manner. No chronic venous congestion was seen. Liver tissues were normal without any degeneration and destruction. No fatty change was found in the liver. Sinusoidal dilation appeared normal. The intestinal mucosa was observed to have numerous intact villi, throughout its length lined by well-defined simple columnar epithelium. Goblet cells were found interspersed among columnar absorptive epithelial cells. Thus, intestinal structure exhibited a normal fashion. The spleen was normal in size; its red and white pulp regions were distinguishable. Hence it is crystal clear that the newly synthesized polymeric carrier is safe for living tissue.

Table 4 Clinical findings for acute in vivo toxicity studies for fabricated hydrogel (PLMA).
Full size table
Table 5 Blood analysis and clinical chemistry report for acute in vivo toxicity studies for fabricated hydrogel (PLMA).
Full size table
Fig. 13
figure 13

Histological examination of different tissues (A) intestine, (B) lungs, (C) heart, (D) right kidney, (E) left kidney, (F) liver, (G) stomach, (H) spleen of rabbits of group I (control) and group II (test–treated with PLMA hydrogel).

Full size image

Conclusion

Main objective of recent study was to load Famciclovir in newly synthesized hydrogel to have controlled delivery of drug for an extended period thereby reducing its dosing frequency. Through this project, we have successfully loaded Famciclovir in a newly developed hydrogel consisting of numerous ratios of polymer (Pluronic F127), monomer (MAA), cross-linker (MBA), and initiator (APS). Polymeric nexus has been developed by using free radical polymerization technique. Developed PLMA hydrogel exhibited numerous promising properties including pH-dependent response, fluid imbibition, drug encapsulation, and release at intestinal pH. Formulation PLMA-4 having 5 g of MAA showed the highest swelling ratio and enhanced Famciclovir loading and release at pH 7.4. In vitro dissolution studies conducted on Famciclovir-loaded PLMA hydrogels confirmed the controlled release of the drug for an extended time which can help in reducing the dosing frequency of drug. This can also help in maintaining desired serum drug levels for a prolong time. Similar release studies for Famciclovir marketed tablet showed inferior results. PXRD, FTIR, SEM, TGA–DSC techniques were employed on hydrogel and results revealed successful loading of Famciclovir in Pluronic F127 and MAA nexus. Also, the crystallinity of Famciclovir got converted to an amorphous one upon loading in PLMA hydrogel as revealed through PXRD. It was found that swelling has a direct relation with the release of drug. DD Solver application on entire nine formulations unveiled the following of zero order and Korsmeyer-Peppas release models. First order was followed by commercially available conventional tablet of Famciclovir. Oral acute toxicity studies executed on rabbits certified the safe and non-toxic nature of polymeric combination. The whole picture declares that the fabricated system can be employed as promising carrier for Famciclovir. This polymeric network being loaded with Famciclovir can help in improving therapeutic outcomes as it reduces the dosing frequency of the drug. Developed formulations can help in controlled and targeted delivery of drugs for viral diseases. It is being designed for site-specific drug delivery and release. It is also supposed to mask the side effects associated with Famciclovir especially related to abdominal issues including GIT upset and nausea. Further research including in vivo pharmacokinetic studies (plasma level of drug at different time intervals, bioavailability, half-life), comparative in vivo anti-viral activity of PLMA loaded hydrogels with conventional dosage forms, long term, chronic safety studies are proposed for this drug delivery carrier for advancement in treatment options regarding Herpes Simplex and Varicella Zoster infections. Additionally, immune response evaluation including cytokine profiles are also recommended. Our study design and outcomes will pave the way for these activities in recent future.