Citrus aurantifolia -derived carbon quantum dots with red fluorescence emission for codelivery with curcumin as theranostic liposomes for lung cancer

Background Carbon dots (CDs) derived from Citrus aurantifolia represent a promising platform for advanced cancer therapy and diagnostics (theranostics). These CDs are synthesized through a sustainable and cost-effective hydrothermal method, utilizing fruit juice as a green carbon source. Despite the potential, research on the synthesis of citrus-based CDs, especially regarding their red fluorescence emission, which is crucial for enhanced tissue penetration and biomedical efficacy, remains limited. Results In this study, CDs were successfully synthesized from C . aurantifolia fruit, yielding nanoparticles below 5 nm in size (PDI 0.231 ± 0.04). Characterization revealed favorable optical properties, including excitation-dependent fluorescent behavior with prominent red emission under higher excitation wavelengths, a quantum yield of 8.17%, and stable photoluminescence. Chemical composition analysis using XPS, FTIR, and XRD confirmed the purity and structure of the CDs. To explore their biomedical application, CDs were co-loaded with curcumin into liposomes. The formulations had a mean size of 177.2 ± 3.6 nm (PDI 0.270 ± 0.012), demonstrated efficient drug entrapment (60.32 ± 2.24%), and exhibited rapid release kinetics, with 90.21 ± 2.16% of the drug release within 8 h. In vitro studies using A549 lung cancer cells demonstrated superior cellular uptake and cytotoxicity of Cur-CD-loaded liposomes compared to curcumin alone (Cur-Suspension), achieving IC50 values of 0.093 ± 0.011 µg/ml and 0.016 ± 0.006 µg/ml, respectively. Conclusion This research underscores C . aurantifolia as a viable natural source for green CD synthesis. The obtained CDs with red fluorescence emission, optimized through reaction conditions and excitation wavelengths, hold promise for enhanced biological applications, particularly in the realm of lung cancer therapy. The findings advocate for further exploration and refinement of citrus-based CDs as versatile theranostic agents, capitalizing on their sustainable origins and potent biomedical properties. The combination of citrus-derived CDs with curcumin loaded into liposomal formulations represents a potent theranostic strategy for lung cancer treatment, leveraging the unique properties of CDs and their potential for targeted and effective therapy.


Background
Lung cancer is one of the leading causes of cancer-related mortality worldwide.Currently, lung cancers are treated with a mix of chemotherapy, surgery, and radiation therapy, according to the kind and stage of cancer [1].However, diagnosis of the disease at an early stage is rare.Consequently, treating the disease with radiation or surgery becomes challenging at advanced stages.Hence, chemotherapy is the only treatment available for lung cancer diagnosed at an advanced stage.However, despite various efforts we still lack a suitable delivery system for sufficient delivery of chemotherapeutic agents to the cancerous region in lungs with reduced toxicity.Nanoformulations have been proposed as the most potential solutions to these challenges [2].In recent years, liposomes have become the most clinically established systems for delivering therapeutics to lung cancer patients in advanced stage, and they have the potential to be a more effective approach to combat lung cancer [3,4].Despite significant advancements, the success rate of treating lung cancer with liposomes remains limited due to several issues, including potential toxicity, lack of specific targeting, and challenges in drug disposition [5].Hence, there is need for a more multifunctional theranostic approach for efficient delivery in lung cancer by loading both imaging agents and therapeutics agents enabling for early diagnosis and site-specific drug delivery [6].CDs can be potential theranostic systems for lung cancer and have shown promising results.CDs can work in the nano-bio interface, where the light induces specific photochemical or photophysical reactions allowing diagnosis and chemotherapeutic delivery.They can also be used in cytotoxic cancer therapy by photodynamic and photothermal effect [7].
CDs are promising fluorescent nanomaterials of size less than 10 nm.Besides having the common properties of nanomaterials, these CDs are considered safer, with excellent photostability, good biocompatibility, are easy to functionalize, and have low cytotoxicity.Due to their properties, including eco-friendliness and low cost, CDs are widely used in various biomedical applications such as ion detection, nanomedicine, targeted delivery, bioimaging, biosensing, and diagnostics [8].CDs because of its size have advantage of better site-specific delivery and due to its fluorescence properties have imaging property that may help in a more theranostic design for lung cancer therapy.These CDs can be prepared from renewable biomass without toxic reagents by sustainable methods of green synthesis and therefore have the advantage of abundant and low-cost sources [9][10][11].Different natural carbon sources such as watermelon peel, mango fruit, food caramels, egg yolk, banana, linseed, milk have been used [7].Green synthesis has recently been explored for its sustainable technology, and hence, CDs prepared by these safe techniques are more preferred.Moreover, the use of natural carbon sources such as biowastes makes the process very environmental-friendly, safe, and sustainable.The CDs can be prepared by both 'top-down' and 'bottom-up processes.' Top-down process generally requires long processing times, harsh reaction conditions, and expensive materials and equipments.In comparison, bottom-up approach is more preferred in terms of nontoxicity, simple reactions, cost-effectiveness, and eco-friendly [12].Among different methods of bottomup preparation, the hydrothermal method is most widespread in use for synthesizing fluorescent CDs from safe and nontoxic biomass [13].The hydrothermal method of synthesis is better than other methods because of its low cost, nontoxicity, environment-friendly, and higher quantum yield [14].Such an environment-friendly method of synthesis is necessary to reduce the generation of toxic and undesirable by products like the other methods [15].The method has good scalability that can be further increased by producing in continuous system using continuous flow microreactor for large commercial production [8].Microwave synthesis, which operates on the same principle as hydrothermal synthesis, offers efficient scale-up by reducing synthesis time.However, this method is limited because of the production of fluorescent impurities.Recently, hydrothermal carbonization of natural carbon source from aloe, bamboo leaves, pork, coffee beans, berries has been successfully applied for green synthesis of fluorescent CDs [16].Phytochemicals like flavones, flavanones, flavonoids, limonoids, and triterpenoids are reported to play a vital role in producing the ultra-small CDs.Citrus aurantifolia has been reported to be rich in these phytochemicals and can be a very potential source for such green synthesis; however, studies on CDs based on this fruits are limited and need to be explored [17].Most of the carbon dots emits blue or green fluorescence that restricts its use in bioimaging due to poor penetration.Hence, there is need to develop reliable approaches for preparing long-wavelength (600-800 nm) red photoluminescence (PL) emission CDs with narrow-band luminescence.These are more sought for biomedical applications due to low light absorption, weak auto-fluorescence, and deep tissue penetration [18].The excitation wavelength-dependent red shifts in PL (photoluminescence) emission is advocated for achieving multicolor imaging besides change in reaction condition and source of carbon [19].
Curcumin is a polyphenolic compound with anticancer potential.It acts on a wide spectrum of molecules like NF-jB, Cox-2, AP-1, and MAPK (mitogen-activated protein kinases) signaling components and has apoptotic effect [20,21].Unlike several other chemotherapeutic drugs, it is safe and well tolerated even at high doses in humans [22].Its anticancer activity has been demonstrated against cancers of various origins [23][24][25][26][27] including lung cancer cell lines [28].However, its use is still limited due to its low water solubility, which results in poor absorption and bioavailability [29].Liposomes can be a potential system for improving its cellular absorption and thereby cytotoxicity.They have advantages in delivery poorly soluble drugs, in targeted drug delivery, reducing the toxic effect of drugs, extending circulation half-life, altering tissue distribution, thereby enhancing the therapeutic index [30].The CD-loaded liposomes shall be a theranostic design with the fluorescence of the CDs for imaging and the liposomes being used for the efficient delivery of the chemotherapeutic drug as well as CDs imparting a photothermal therapy.
In the present study, we have therefore designed CDsloaded liposomes for a more theranostic delivery of curcumin for lung cancer.These theranostic systems shall be efficient design for imparting a synergistic effect due to the photothermal effect of the CDs and chemotherapeutic effect of curcumin, besides the imaging property of the fluorescent CDs.The CDs were obtained by sustainable green synthesis method by hydrothermal carbonization using the C. aurantifoliajuice as carbon source.Optical and physicochemical properties of the CDs and the effect of the process variables were thoroughly studied with more emphasis on achieving sufficient red fluorescence emission for better efficacy.The CD-Curcumin-loaded liposomes were characterized and evaluated for their efficiency, drug delivery, stability and in vitro cytotoxicity, and uptake in A549 lung cancer cells.

Materials
Citrus aurantifolia fruits were collected locally from Guwahati, Assam, India.Cholesterol, soya lecithin, and chloroform were purchased from the SISCO laboratories Pvt. Ltd. (Mumbai, India), methanol was purchased from Changshu Yangyuan Chemical Co. Ltd. (Suzhou, Jiangsu, China), HPLC grade methanol was purchased from Spectrochem Pvt. Ltd. (Mumbai, India), water for HPLC was purchased from Sisco Research laboratories Pvt. Ltd. (Maharashtra, India), and gradient grade acetonitrile for HPLC was purchased from Merck (Mumbai, India).
Human pulmonary cell lines A549 were obtained from NCCS Pune, India.Ham's F-12 K medium and Pen-Strep antibiotics were purchased from the HiMedia Lab Pvt Ltd. (Mumbai, India).Fetal bovine serum (FBS) was obtained from GibcoInc, USA.

Green synthesis of CDs
CDs were prepared by hydrothermal method from fresh juice of C. aurantifolia used as a natural source of carbon precursor.Briefly, 30 ml of the filtered juice was taken in a Teflon-lined stainless steel hydrothermal autoclave (Technistro 200 ml) and heated for 5-8 h at high temperature (160-220 °C).After completion of the hydrothermal carbonization reaction, the autoclave was allowed to cool down to normal room temperature and the carbonized solutions obtained were then centrifuged at 10,000 rpm for 15 min to get rid of the large particles and filtered the supernatant with a 0.22-μm membrane in order to get pure CDs.The obtained CDs solutions were stored at 4 °C for further studies [22].

Optimization and characterization of the CDs
The CDs prepared by the above-mentioned method were optimized by changing the temperature of heating, duration of autoclaving, and pH [28].Temperature was changed from 160 to 220 °C, the duration time was changed from 5 to 8 h of heating, and the pH was varied from 3 to 6 in acidic range and in alkaline pH.The extent of carbonization was studied from its effect on size, yield, fluorescence intensity, and UV absorbance.The optimized CDs were prepared and lyophilized for further characterization.

Optical properties of the CDs
The optical properties of CDs such as absorption, photoluminisecnce, and phosphorescence are important fundamentally as well in terms of its application.UV (ultraviolet) absorbance spectra, fluorescent spectra, decay, and quantum yields of CDs were therefore explored to access these optical properties of the CDs [31].
UV spectrum UV absorption spectra were measured using a UV-1900i UV-Vis Spectrophotometer (Shimadzu, Japan) in wide range from 200 to 800 nm to study the effect of the hydrothermal reaction and change in parameters on the absorbance pattern.

Fluorescence intensity
The fluorescence intensities at different excitation wavelengths from (550-820) nm and at different pH (3)(4)(5)(6)(7)(8), temperature, and duration of synthesis were observed to study how these parameters affect the luminescence intensity of the CDs.The fluorescence intensity was detected on the RF-6000 spectrofluorophotometer (Shimadzu, Japan).
Determination of quantum yield (QY) Quantum yield of obtained CDs solution was determined using quinine sulfate (0.1 M H 2 SO 4 QY = 0.54 at 360 nm excitation) as reference.QY of the CDs was calculated by the following equation [25]:

Transmission electron microscopy (TEM)
The structure, shape, and morphology of the prepared CDs were studied using TEM.The CDs stained with 1% w/v phototungstic acid after transferring to copper grid coated with carbon.TEM pictures were then obtained using TEM (JEM-2100 PLUS (HR), Jeol).

FTIR (Fourier transform infrared) study
The FTIR analysis was performed to identify the surface functional groups of the prepared CDs.The lyophilized CD was analyzed using FTIR (BRUKER, ALPHA-E) to obtain the transmittance peak for the CDs [32].

X-ray photoelectron spectrometer (XPS) study
XPS study was performed to elucidate the surface elemental composition of the CDs and to access the structure of the CDs.The carbon, nitrogen, and oxygen content was determined in the CDs using Thermo Fischer Scientific X-Ray Photoelectron Spectrometer (Escalab Xi+) equipped with a monochromatic Al K alpha source.Pass energy of 50 eV was used for the analyses with a200eV survey.

XRD (X-ray diffractometer) analysis
XRD determination was performed to check the crystallinity of the CDs with a Phillips X'Pert Pro Powder X-ray Diffractometer (XRD) having a diffracted beam monochromator, using Cu radiation.Intensity of diffraction was observed at an angle of 2θ from 10 to 90° maintaining step size of 0.02° and time of 1 s.A voltage of 40 kV and 30 mA current were maintained by the voltage and current generator, respectively, during the recording.

PL decay and stability
The fluorescence intensities of the optimized CDs were observed for a period of One month for any change in the fluorescence intensity.The fluorescence intensity at 820 nm was observed for any such PL decay.The physical stability of the CDs was also checked by storing in glass vials at room temperature and observed for any physical change in size or appearance.

Preparation of liposomes
Thin-film hydration method was used for preparation of the liposomes using a rotary evaporator [32,33].Required quantities of cholesterol and soya lecithin were dissolved in a methanol and chloroform solvent system along with the drug and stirred for 30 min.The organic phase was then evaporated by means of rotary evaporator at 40 °C to produce a thin film of the lipids.The film was then hydrated at the same temperature with a mixture of phosphate buffer solution (PBS) and CDs solution in 1:1 ratio.The mixture was shaken in a rotary shaker (REMI) for half an hour to obtain the liposomal vesicles.

Vesicle size
The vesicle size and homogeneity of the CDs-loaded curcumin liposome were determined by Zetasizer, Malvern Nano S90, using dynamic light scattering after diluting in 1:20 ratio.All observations were done at room temperature by scattering at 90° angle.The zeta potential of the formulation was determined using Zetasizer ver.V 2 2 Malvern.

HPLC (high-performance liquid chromatography) method development
The optimized HPLC method as reported in our earlier work [34] was used for determination of curcumin.The method involved uses of acetonitrileacidic water (pH3 adjusted with acetic acid) 80:20 ratio at flow rate of 1.0 ml/min.The run time was 10 min, and the determination was done at 425 nm wavelength.The determinations were performed in HPLC (Waters Arc) system using a reversed-phase C18 Column.

Determination of entrapment
The entrapment of curcumin was observed in the CD-Cur-Liposome formulation.0.5 ml of CD-loaded curcumin liposomal formulation was centrifuged at 15,000 rpm for 10 min in a cooling centrifuge machine (REMI).The amount of unentrapped drug was determined by measuring the absorbance of the collected supernatant in UV-Vis spectrophotometer (SHIMADZU, UV-1900).The entrapment efficiency was then determined using the following equation [33]:

Stability study
Physical stability of the liposomal formulation was observed in terms of size, PDI, and fluorescence intensity, for 3months at 4 °C, room temperature, and 40 °C in both dark and bright conditions.

MTT(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) study in A549 cell lines
The human lung cancer cell lines A594 cells were cultured in Ham's F-12 K medium (10% fetal bovine serum) with penicillin and streptomycin as antimicrobials and incubated at 37 °C under 5% CO2, 95% air, and 95% humidity.Cell viability was determined by MTT analysis.1.8 × 10 6 cells/well were seeded into 96-well plates and allowed to attach to the well surfaces by incubating for 2 h.After that, the CD-Cur Liposomes and CUR-Suspension treatments were given in triplicate and incubated for 24 h.The MTT solution was then added, and the culture was maintained at 37 °C for an additional 4 h.DMSO (200 μl) was added to dissolve the formazan crystals after removing the supernatants, in each well.The absorbance in the wells was then measured at 570 and 655 nm (for background correction) using a microplate reader (Agilent 800ES) [35].The % cell viability was calculated using the equation: The % cell viability was then plotted into a graph, and the IC50 value was determined.

In vitro uptake study
In vitro uptake of CD-Cur liposomes and CUR-Suspension against A549 human lung cancer cells was determined by fluorescent microscope (XD-RFL Fluorescence Microscope, SDPTOP, China).Briefly, cells were cultured at a density of 2 × 10 5 cells/ml in 12-well plate and incubated overnight at 37 °C for 24 h in a CO 2 incubator.These were then spent washed with 1 ml of 1X PBS.The cells were then treated with required concentration of the formulations in 1 ml of culture medium and incubated the cells for 2, 4, or 6 h.The cells were washed two times with 1X PBS and then 500 μl mounting medium was added before imaging.The cells were then observed using fluorescence microscopy under objective of 20X with filter cube having excitation 470/40 and emission 525/50, and the images were analyzed using ImageJ Software v1.48.The cells without any CDs treatment were used as control for comparison [13].

Green synthesis of carbon quantum CDs
CDs were successfully prepared by hydrothermal method by using fresh juice of C. aurantifolia as a natural source of carbon precursor.Following carbonization, it was observed that over time, the particle size of the black colored CDs solution decreased as it reached equilibrium.Spherical CDs were synthesized, resulting in particles with a size 3 ± 2 nm and narrow size distribution, indicated by a PDI of 0.231 ± 0.04 as shown in Fig. 2a.

Optical properties of the synthesized CDs
The CDs prepared by the above-mentioned method were optimized by changing the temperature of heating, time of autoclaving, and pH.The temperature was varied from 160 to 220 °C, and the duration time was varied from 5 to 8 h of heating, and the extent of carbonization was studied from its effect on size, yield, fluorescence intensity, red fluorescence emission, and UV absorbance (Fig. 1a, b, respectively).From the study, the CDs obtained at 8 h were found to be most suitable based on the size and fluorescence intensity of the obtained CDs.Autoclaving temperature at 220 °C was found to be optimized based on the effect of the temperature on the yield, type, and intensity of fluorescence.The size of the CDs decreases with increase in temperature and time of autoclaving.Similarly, it was observed that the fluorescence intensity increases with increase in temperature and time of autoclaving.The fluorescence intensity of the CDs at different pH (3)(4)(5)(6)(7)(8) was also observed, as pH is a major factor affecting the fluorescence intensity of the CDs.It was observed that with increasing pH the intensity decreases with a blue shift toward shorter wavelength and acidic pH was more essential for better fluorescence intensity.The results are shown in Fig. 1c.Hence, autoclaving temperature at 220 °C for 8 h in acidic pH was considered to be the optimized condition for the green synthesis of CDs with sufficient fluorescence emission from C. aurantifolia fruit juice as shown in Table 1.
The CDs exhibited stable fluorescence intensity with excitation-dependent fluorescence emission.The CDs exhibited green to blue fluorescence at excitation wavelengths in the UV region.However, at higher excitation wavelengths, significant red fluorescence emission was observed in the 650-820 nm range.3D Spectrum of the CDs shows excitation-dependent fluorescence emission with considerable red fluorescence at higher excitation wavelength as shown in Supplementary File 1. Similar results have been reported in Wang et al. [36], Kong et al. [37].When illuminated under normal light, the synthesized CDs solution was yellowish in color; however, it exhibits intense blue-red luminescence under longer excitation wavelengths.The CDs showed broad absorption from 250 to 400 nm, showing shoulder peak around 301-304 nm.The optimized CDs were prepared and lyophilized for further characterization.

Determination of fluorescence quantum yield
Quantum yield of the CDs solution was determined by using quinine sulfate (0.1 M H 2 SO 4 QY = 0.54 at 360 nm excitation) as reference.The fluorescence intensity value of CDs (F CDs) was found to be 21,140 at 360 nm excitation wavelength, and fluorescence intensity value of quinine sulfate in 0.1 M H 2 SO 4 (F r ) was found to be 123,741 at 360 nm excitation.Similarly, the UV absorbance values of CD solution (A CDs ) and quinine sulfate (A r ) were found to be 0.0431 for CDs and 0.0382 for quinine sulfate.The quantum yield was obtained as 8.17% which is similar to other reported works from different sources.

TEM studies
The structure, shape, and morphology of the prepared CDs were observed by using TEM (JEM-2100 PLUS (HR), Jeol).The results, as depicted in Fig. 2b, reveal that the CDs are uniformly dispersed and possess a round shape.Their diameter falls within the range of 1-5 nm, with an average size of 3 ± 2 nm.The CDs showed stacking to form bigger structures.

FTIR study
The FTIR analysis was done to identify the surface functional groups of the prepared CDs.It was observed that the transmittance peak of the synthesized CDs exhibited characteristic peaks at 2932.66, 2601.71,1393.62,1701.16, 1182.35, 1034.26, 878.11, and 783.98 cm −1 .These spectral peaks suggest the synthesis of CDs with versatile organic groups on its surfaces.The results are shown in Fig. 3a.

XPS studies
X-ray photoelectron spectrometry (XPS) was studied to investigate the surface functional groups of the CDs.The XPS spectrum of the CDs exhibits 3 peaks each at 288, 401, and 535 eV, which are ascribed to C1s, N1s, and O1s signals, respectively.The result, illustrated in Fig. 3b, demonstrated the composition of the CDs, revealing the presence of carbon, nitrogen, and oxygen elements.

XRD analysis
XRD analysis was carried out to assess the crystalline nature of the CDs.The analysis showed a broad diffraction peak at 2θ = 20° as shown in Fig. 3c.This indicates reduced crystallinity or amorphous nature of the CDs [27,38].

PL decay and stability
Observations revealed that the emission intensities of the CDs remained nearly constant for up to 30 days,

Preparation of liposome
Homogenous liposomes were obtained by the thin-film hydration method using cholesterol and soya lecithin in methanol and chloroform as the organic phase for formation of lipid film and hydrated by phosphatebuffered solution (PBS) with PVA 0.5%.Lipids ratio of 85:15 and solvent ratio of 20:10 were considered as optimized conditions based on the observed size and homogeneity of the liposomal vesicles.The CDs-loaded liposomes were prepared by replacing 5 ml of PBS with 5 ml of CDs solution as the hydrating system.Curcumin was added along with the lipids dissolved in the organic phase in order to get Curcumin and CDs-loaded multifunctional liposomes.

Particle size
The prepared liposomes had an average particle size of 177.2 ± 3.6 nm and a PDI of 0.270 ± 0.012, as shown in Table 2.The average particle size remained unchanged on preparation of CDs-loaded liposomes showing size and PDI of 207.3 ± 7.2 nm and 0.153 ± 0.064, respectively.The size and dispersity were better when co-loaded with Curcumin and CDs.Zeta potential revealed that the CD-Liposome was negatively charged (− 5.83 mV), indicating the dispersed particles in the suspension are negatively charged.The UV absorbance and fluorescent intensities of the prepared liposomes were observed.Comparative analysis of the blank liposomes, CDs,   observed with the CD-Curcumin-co-loaded liposomes exhibiting maximum fluorescence intensity at 630 nm rather than 820 nm as in case of CDs alone.The result is shown in Fig. 6.

Drug entrapment efficiency
The drug entrapment efficiency of the optimized Curcumin-CD-loaded liposomes was found to be sufficient with 60.32 ± 2.24%.The entrapment was found to be dependent on the lipid content, solvent system (ratio) and preparation time.

In vitro drug release study
The drug release of the Curcumin-CD-loaded liposomes was studied by dialysis membrane bag technique using PBS-7.4+ 0.5% tween80 as release medium.From the HPLC analysis of the aliquots using the optimized method, it was found that 90.21 ± 2.16% of the drug was released in 8 h.

Stability study
The physical stability of the liposomal formulation was observed in terms of size, PDI, and fluorescence intensity for 3 months at 4 °C, room temperature, and 40 °C in both dark and bright conditions.The Cur-CD-loaded liposomes were found to be stable in dark conditions; however, some degradation and formation of particulate matter were observed on storage under light exposure.It was also observed that PVA (polyvinyl alcohol) 0.5% was essential to maintain the physical stability of the liposomes as increase in size distribution was observed in liposomes without PVA in room temperature.

In vitro cytotoxicity study
Cell viability of free curcumin suspension and Curcumin-CD-loaded liposomes was evaluated using MTT assay on human A549 lung cancer cell line.Different concentrations of free curcumin suspension and Curcumin-CD-loaded liposomes (ranging between 10 and 300 ng/ml) were used for the study.Both suspension and liposomes showed concentrationdependent cytotoxicity as shown in Fig. 7. IC 50 values were determined after 24 h of incubation and found to be 0.093 ± 0.011 µg/ml and 0.016 ± 0.006 µg/ml, respectively, as shown in Table 3. Cytotoxicity was assessed in terms of % cell viability, and CD liposomes demonstrated higher viability compared to the suspension.

In vitro cell uptake studies
The in vitro cell uptake studies using fluorescent microscope showed sufficient uptake in treated A549 cells.The uptake in cells treated with Cur-CD-loaded liposomes was comparatively higher compared to Cur-Suspension.The extent of uptake increased from 2 to 6 h post-treatment and with highest uptake was observed at 6 h in both the treatments.The comparative results are shown in Fig. 8.

Discussion
CDs were successfully prepared by hydrothermal method by using fresh juice of C. aurantifolia as a natural source of carbon precursor.Citrus aurantifolia contains certain active phytochemicals like flavones, flavanones, flavonoids, limonoids, and triterpenoids.These phytomolecules play a vital role in producing the ultrasmall CDs [16].Formulated CDs exhibit fluorescence intensities across various wavelengths, ranging from 550 to 800 nm.The resulting fluorescence behavior may be attributed to the method of synthesis and the starting material as both these factors play important role in the CDs properties [39].The CDs showed red fluorescence with good intensity at 820 nm.Generally, CDs emit green or blue fluorescence; however, there is an increased demand for red fluorescence.Achieving red fluorescence (600-800 nm) is an urgent need as these CDs are considered to have several advantages for biological applications, particularly due to their deep tissue  penetration and reduced problems of auto-fluorescence [17].It was observed that the intensity increases with increasing excitation wavelengths revealing that the CDs fluorescence is strongly dependent on the excitation wavelength [22].Since the PL of the CDs can be regulated by the excitation wavelength, hence these resulting CDs by the optimized method of green synthesis, emitting red fluorescence without chemical treatment, hold significance and may result in better efficacy.The optical behavior may also be attributed to the absorption behavior.CDs show strong absorption at appropriate band gap exhibiting fluorescence.The absorption behavior in CDs depends on the method of synthesis and the carbon sources which affect the content, type of surface groups, and size of π-conjugated domain [12].The CDs showed broad absorption from 250 to 400nm that can be ascribed to π-π* of aromatic C=C bonds and showed shoulder peak at around 301-304 nm possibly caused by the n-π* transition of the amine or carbonyl groups on the surface of the CDs or other connected groups [35].Moreover, the surface functional groups in these CDs may also have effect on these absorption bands [39].The absorption band also showed tail in the visible region resulting in red fluorescence emission which can be attributed due to the presence of π-conjugated electrons in the sp 2 domain or the surface groups [32].
The reaction conditions, such as temperature, time, and pH, are very essential for synthesizing CDs with optimum optical and chemical properties [28].Hence, in the study, the CDs prepared by the above-mentioned method were optimized by varying the temperature, pH, and autoclaving time.Temperature is the most important factor for hydrothermal reaction as the reaction involves loss of water from hydrates and graphitization during the process [28]; hence, the temperature was varied from 160 to 220 °C.Increasing temperature in lower temperature range enhanced the initial carbonization rate; however, at higher temperatures it decreased the yield.Higher temperature results in higher quantum yield due to the effective conversion of natural constituents into carbonization.Depending on the hydrothermal temperature, the emission peaks range from the blue-green region to the infrared region [40].The different temperature also affects the size of the CDs due to the different extent of degradation of the precursor resulting in nucleation at different conditions.From the study, autoclaving temperature at 220 °C was found to be optimized.
The reaction time being another important factor was varied from 5 to 8 h of heating, and the extent of carbonization was studied from its effect on size, yield, fluorescence intensity, and UV absorbance.The fluorescence intensity and yield of the CDs were found to increase with reaction time under autoclaving.This suggests that the increased reaction time might assist in better and complete carbonization and might be essential for the formation of CDs.The complete carbonization due to longer reaction time might result in increase in the intensity of spectra which may further lead to increase in quantum yield.Similar results have been reported in other works [40].
From the study, the CDs obtained at 8 h were found to be most suitable.Additionally, it was observed that fluorescence intensity increased and size decreased after overnight storage compared to freshly prepared CDs.This suggests some degree of passivation behavior following the carbonization reaction, which may have contributed to the increased fluorescence [41].However, no external chemical treatment was made for this passivation.The above behavior might also be due to the reduced size of CDs [42].
Similarly, pH is another important parameter that affects the yield and luminescent intensity as the route of reactions in different pH conditions affects these parameters and the extent of carbonization.It was observed that with increasing pH, the intensity decreases and shifts toward lower wavelength and acidic pH was more essential for red fluorescence emission.In an acidic environment (pH 3-6), the emission wavelength showed sufficient red fluorescence.The photoluminescence pH sensitivity of CDs is dependent on reaction conditions, synthetic methods, and raw materials.The present study showed increase PL intensity at acidic pH that might be due to the reversible protonation and deprotonation of the CDs surface functional groups [43].
However, the XRD spectra of the samples in different pH conditions presented the same peaks and intensity indicating that the pH does not affect the crystallinity profile of CDs.Similar results have been reported earlier by Lei et al. [44].
Quantum yield of obtained CDs solution was measured by using quinine sulfate (0.1 M H 2 SO 4 QY = 0.54 at 360 nm excitation) as a reference.High quantum yield at 8.17% was obtained that might be due to presence of high acidic (citric acid, tartaric acid, and ascorbic acid) and neutral constituents (carbohydrate) in the plant extract [42].The results demonstrated stable and strong photoluminescence that depended on the excitation wavelength.The quantum yield generally depends on the carbon sources, method of synthesis, and the post-passivation process.The higher quantum yield may be attributed to the source used in the study, the bottom-up process of synthesis, and the effect of temperature, reaction time, and pH as discussed above.Moreover, the overnight storage also resulted in passivation further increasing the photoluminescence.
The structure, shape, and morphology of the prepared CDs were observed by using TEM.The result shows that the CDs are spherical in shape with a diameter range of 1-5 nm.Synthesized CDs exhibited a tendency to aggregate, leading to the formation of larger particles.This behavior could be attributed to the elevated temperatures causing the smaller particles to aggregate and form larger particles with varying sizes.Similar behavior has been reported earlier by Ahmadian-Fard-Fini et al. [38].
The FTIR analysis was done to identify the surface functional groups of the prepared CDs.It was observed that the transmittance peak of the synthesized CD exhibited highest transmittance.Peak at 2932.66 cm −1 corresponds to C-H group stretching vibration which may be due to presence of carbohydrates in the juice [45].The peak at 2601.71 cm −1 corresponds to (-COOH, O-H stretching), and Peak at 1393.62 cm −1 corresponds to the COO group [27].Peak 1701.16 cm −1 results from the vibration absorption of free carboxylic acid [23], 1182.35cm −1 corresponds to -C-O-C functional groups [17], 1034.26 can be attributed to the C-O band [21], and 878.11 and 783.98 cm −1 correspond to aromatic C-H bending which can be attributed to the presence of polyphenols in the juice [45].These spectral characteristics strongly indicate the formation of CDs with versatile organic groups on the CDs surfaces [39].These functional groups are mostly polar in nature that may assist in solubility [28,45].
The XPS spectrum of the CDs exhibits 3 peaks ascribed to C1s, N1s, and O1s signals.These outcomes align with the findings from the FTIR spectra, which suggest that the predominant functional groups within the CDs consist of carbon, oxygen, and nitrogen [13,24].The first and main contribution at 288.5 eV can be assigned to graphitic carbon atom and to the presence of COOH in particular.The weak peak at 401.eV can be assigned to N-H bonds present in the CDs.The peak at 535 eV associated with oxygen corresponds to COOH/OH.XRD analysis was performed to check the crystallinity which showed a broad peak at 2θ = 20°.This suggests the reduced crystallinity or the amorphous nature of the prepared CDs indicating a graphitic nature with highly disordered carbon atom [12,13,27].The XRD analysis shows abroad peak at 2θ = 20° due to 002 planes of graphite.The broad peak and the high interlayer distance indicate the amorphous nature of the CDs due to the oxygen containing functionalities [46].
It was observed that the emission intensities of the CDs remained nearly constant for duration of up to 30 days indicating their impressive stability.This characteristic augments their potential for future application in the field of bioimaging technology [39].
Homogenous liposomes were obtained by thin-film hydration method using cholesterol and soya lecithin in methanol and chloroform as the organic phase for formation of lipid film and hydrated by phosphatebuffered solution (PBS).The liposomes were optimized based on different parameters (evaporation time of organic solvent, ratio of solvent system, ratio of phospholipid to cholesterol).The prepared liposomes had an average particle size of 177.2 ± 3.6 nm and a PDI of 0.270 ± 0.012, which is suitable for targeted drug delivery and extended circulation [47].The size and uniformity were better when co-loaded with Curcumin and CDs.The particle size of the liposomes depended on lipids ratios and solvent ratios.Lipids ratio of 85:15 and solvent ratio as 20:10 were found to give optimized size distribution.The UV spectra showed no significant change in the absorption on loading of CD in liposomes and CD-Curco-loaded liposomes showing shoulder peak around 301-306 nm.However, in the fluorescent emission studies, a blue shift was observed in the CD-Cur-co-loaded liposomes in comparison with CDs indicating some extent of quenching effect due to the lipids present in the liposome.This may also due to the inherent fluorescent properties of curcumin.The drug entrapment efficiency of the Curcumin-CD-loaded liposomes was obtained at 60.32 ± 2.24%.The sufficient entrapment efficiency might be due to the high lipophilicity of Curcumin that helped in its easier entrapment in the lipid layers [48].The higher entrapment of the formulation might also be due to the long hydrocarbon chains of the phospholipid (SPC) that have been used in higher amounts (85:15) in the formulations.High content of lipids might result in better vesicles formation.There are reports suggesting that SPC has a tendency to accommodate higher amounts of drugs [49].However, further efforts to increase the entrapment may be required.The drug release study of the prepared formulation was done by using dialysis membrane bag technique using PBS-7.4+ 0.5%Tween80 as release medium.From the HPLC analysis of the aliquots, it was found that 90 ± 2.16% of the drug was released in 8 h.The liposomes showed initial burst release which might be due to the release of the drug on the surface.The physical stability of the liposomal formulation was observed in terms of size, PDI (polydispersity index), and fluorescence intensity for 3months at 4 °C, room temperature, and 40 °C in both dark and bright conditions.The Cur-CD-loaded liposomes were found to be stable in dark conditions; however, some degradation and formation of particulate matter were observed on storage under light exposure.It might be due to the degradation of curcumin on continued exposure to light as it is photosensitive.Hence, the liposomes need to be stored in light protected containers or dark conditions where it was found to be stable for 3 months.
The in vitro cell uptake studies using fluorescent microscopy showed greater uptake in cells treated with Cur-CD-loaded liposomes in comparison with Cur-Suspension.The extent of uptake increased from 2 to 6 h post-treatment, with highest uptake at 6 h in both the treatments.This increase in uptake in Cur-CD liposomes may be due to liposomal vesicle size, composition, and uptake mechanisms [47].This difference in the cellular uptake might have resulted in more cytotoxicity with CD liposomes showing higher reduction in cell viability in comparison with suspension by MTT assay in A549 cells.This enhanced cytotoxicity may also be attributed to the enhanced permeation and retention of the Cur-CD liposomes.The higher uptake of the drug along with the CDs might therefore result in higher site-specific toxicity and photodynamic effect, thereby resulting in a more multifunctional approach for better tumor inhibition.

Conclusion
It may be concluded from the study that CDs emitting red fluorescence can be successfully synthesized by sustainable hydrothermal carbonization method using the C. aurantifolia juice as carbon source.The Curcumin-CD-loaded liposomes obtained from the process may enhance the cellular uptake of the drug and result in better cellular toxicity.This along with the photodynamic effect of the CDs may result in better tumor inhibition and thereby better therapy.Hence, green synthesisderived CDs-loaded curcuma liposomes can be a promising delivery system for theranostic cure of lung cancer.

Fig. 1 A
Fig. 1 A Fluorescence intensity of CDs at different reaction time.B Fluorescence intensity of CDs at different temperature.C Fluorescence intensity of CDs at different pH.D UV absorbance spectra at different reaction time.E Fluorescent intensity at different excitation wavelength

Formulation
530 ± 1.212 0.231 ± 0.054 CD-loaded liposomes, and CD-Curcumin co-loaded liposomes revealed that all CD-loaded liposomes exhibited a characteristic peak at 300-306 nm, as shown in Fig.5.However, a decrease in absorbance was observed when CDs were loaded into liposomes, with the CD-Curcumin co-loaded liposomes showing the lowest absorbance value, peaking at 0.402 at 301.5 nm.Moreover, a blue shift in the fluorescence emission was

Fig. 2 a
Fig. 2 a Particle size distribution of CDs.b TEM photomicrograph of CDs at 15000X magnification

Fig. 4
Fig. 4 PL decay of CDs on 30th day storage

Fig. 7
Fig. 7 Cytotoxicity study of curcumin suspension and Cur-CD liposome in A549 lung cancer cells

Fig. 8 a
Fig. 8 a Cell uptake studies in A549 cells using fluorescent microscope.(a) Uptake of curcumin suspension at 2, 4 and 6 h.b Cell uptake studies in A549 cells using fluorescent microscope of Cur-CD liposome at 2, 4, and 6 h

Table 3
IC50 values of A549 cells dosed with free curcumin (Cur) suspension and CD-Cur liposome for 24 h