Article

Synthesis of Polyamidoamine Dendrimer for Encapsulating Tetramethylscutellarein for Potential Bioactivity Enhancement Daniel M. Shadrack 1,2 , Egid B. Mubofu 1, * and Stephen S. Nyandoro 1, * Received: 13 June 2015 ; Accepted: 8 October 2015 ; Published: 4 November 2015 Academic Editor: Graeme Cooke 1 2

*

Chemistry Department, University of Dar es Salaam, College of Natural and Applied Sciences, P.O. Box 35061 Dar es Salaam, Tanzania; [email protected] Chemistry Department, St John’s University of Tanzania, P.O. Box 47 Dodoma, Tanzania Correspondence: [email protected] (E.B.M.); [email protected] (S.S.N.); Tel.: +255-784-538-344 (E.B.M.); +255-754-206-560 (S.S.N.); Fax: +255-22-410-038 (E.B.M. & S.S.N.)

Abstract: The biomedical potential of flavonoids is normally restricted by their low water solubility. However, little has been reported on their encapsulation into polyamidoamine (PAMAM) dendrimers to improve their biomedical applications. Generation four (G4) PAMAM dendrimer containing ethylenediaminetetraacetic acid core with acrylic acid and ethylenediamine as repeating units was synthesized by divergent approach and used to encapsulate a flavonoid tetramethylscutellarein (TMScu, 1) to study its solubility and in vitro release for potential bioactivity enhancement. The as-synthesized dendrimer and the dendrimer–TMScu complex were characterized by spectroscopic and spectrometric techniques. The encapsulation of 1 into dendrimer was achieved by a co-precipitation method with the encapsulation efficiency of 77.8% ˘ 0.69% and a loading capacity of 6.2% ˘ 0.06%. A phase solubility diagram indicated an increased water solubility of 1 as a function of dendrimer concentration at pH 4.0 and 7.2. In vitro release of 1 from its dendrimer complex indicated high percentage release at pH 4.0. The stability study of the TMScu-dendrimer at 0, 27 and 40 ˝ C showed the formulations to be stable when stored in cool and dark conditions compared to those stored in light and warmer temperatures. Overall, PAMAM dendrimer-G4 is capable of encapsulating 1, increasing its solubility and thus could enhance its bioactivity. Keywords: PAMAM G4 dendrimer; encapsulation; tetramethylscutellarein; solubilization; in vitro release; stability

1. Introduction The development of biodegradable polymeric nanoparticles for effective drug and gene delivery has significantly increased in recent years [1]. Of these, polyamidoamine (PAMAM) dendrimers have been of significant interest due to their unique properties, which provide an endless application in drug delivery. For instance, they are used for gene therapy, drug delivery and magnetic resonance imaging [2]. The use of dendrimers in drug delivery or carrier system to increase therapeutic index, efficacies and solubility in aqueous media is well reported [3,4]. One of the intrinsic properties that limit the clinical applications of the potentially active natural products and their synthetic analogues is poor water solubility [5]. A number of techniques have been used to increase solubility of sparingly water-soluble compounds, among them, being the drug–dendrimer complexation [2]. The ability of PAMAM dendrimers to encapsulate and complex with drugs and other biologically active natural products is based on their unique properties such Int. J. Mol. Sci. 2015, 16, 26363–26377; doi:10.3390/ijms161125956

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Int. J. Mol. Sci. 2015, 16, 26363–26377

Int. J. Mol. Sci. 2015, 16, page–page

as monodispersity, large surface area, high degree of molecular uniformity, specific size and shape which which are are significantly significantly different different from from their their classical classical linear linear polymer polymercounterparts counterparts [6]. [6]. Thus, Thus, the the drug–dendrimer complexation is is currently currently widely widely used drug–dendrimer complexation used in in pharmaceutical pharmaceutical industries industries as as an an approach approach to to increase increase stability, stability,solubility, solubility,bioavailability bioavailabilityand andcontrolled controlledrelease releaseofofdrugs drugs[2,6]. [2,6]. Flavonoids and other polyphenolic compounds are known to possess Flavonoids and other polyphenolic compounds are known to possess cytotoxic cytotoxic activities activities on on different However, water-insolubility water-insolubility and different cancer cancer cells cells and and have have other other biomedical biomedical potentials potentials [7,8]. [7,8]. However, and slow dissolution rate rate remains remains aa serious seriousdrawback drawbackfor fortheir theirclinical clinicalapplications applications [7,9]. Despite slow dissolution [7,9]. Despite the the plethora of evidence PAMAM dendrimerstotomediate mediatemedical medicalapplication application of of most most natural plethora of evidence on on PAMAM dendrimers natural products, been reported reported on on the the encapsulation encapsulation of of flavonoids flavonoids onto onto PAMAM PAMAM and products, little little have have been and other other encapsulating agents [9–11]. Thus, the essence of undertaking investigations involving PAMAM encapsulating agents [9–11]. Thus, the essence of undertaking investigations involving PAMAM and and a flavonoid 5,6,7-trimethoxy-2-(4-methoxyphenyl)-4H-chromen-4-one a flavonoid 5,6,7-trimethoxy-2-(4-methoxyphenyl)-4H-chromen-4-one (Tetramethylscutellarein (Tetramethylscutellarein (TMScu, 1), (Figure (Figure 1)). 1)). The Thecompound compoundisisknown knownto to exhibit exhibit anti-inflammatory anti-inflammatory properties properties [12] [12] and (TMScu, 1), and moderate cytotoxic activity against human breast cancer cell lines (MCF-7) and anti-tuberculosis moderate cytotoxic activity against human breast cancer cell lines (MCF-7) and anti-tuberculosis potency against Mycobacterium Mycobacteriumtuberculosis tuberculosis(H37Rv (H37Rvstrain) strain) [13], whereas structural analogues potency against [13], whereas its its structural analogues are are well acknowledged for treatment of various ailments [8,14]. Dendrimers effectiveness well acknowledged for treatment of various ailments [8,14]. Dendrimers effectiveness for for encapsulation, encapsulation, solubolization solubolization and and drug drug delivery delivery depends depends on on the the number number of of generations generations and and terminal terminal group Hence, our our investigations investigations explored explored the the capacity capacity of of the group among among other other factors factors [3–6]. [3–6]. Hence, the amine amine terminated PAMAM G4 dendrimer to encapsulate compound 1, a model compound among terminated PAMAM G4 dendrimer to encapsulate compound 1, a model compound among other other bioactive bioactive flavonoids flavonoids [13] [13] for for potential potential bioactivity bioactivity enhancement. enhancement. The The compound compound solubilization solubilization under under the thethe synthesized G4 PAMAM dendrimer, in vitroin release stability the dendrimer the influence influenceofof synthesized G4 PAMAM dendrimer, vitro and release andinstability in the complex were studied, thestudied, results of which areofreported in reported this paper. dendrimer complex were the results which are in this paper.

Figure 1. 1. Chemical of tetramethylscutellarein tetramethylscutellarein (TMScu, (TMScu, 1). 1). Figure Chemical structure structure of

2. Results and Discussion 2. Results and Discussion 2.1. Synthesis and Characterization Characterization of of Polyamidoamine Polyamidoamine (PAMAM) (PAMAM)Generation GenerationFour Four(G4) (G4)Dendrimer Dendrimer 2.1. Synthesis and The synthesis synthesis of of PAMAM PAMAM G4 G4 dendrimer dendrimer was was carried carried out out by by aa divergent divergent approach approach with with reagent reagent The excess, wherein wherein ethylenediaminetetraacetic ethylenediaminetetraacetic acid acid (EDTA) (EDTA) was was used used as as core. core. All All synthetic synthetic steps steps were were excess, completed and confirmed by copper sulfate color chelation reactions. While half generations gave completed and confirmed by copper sulfate color chelation reactions. While half generations gave deep blue color, full generations had purple coloration. The purple color formed was due to the deep blue color, full generations had purple coloration. The purple color formed was due to the reaction of of terminal terminal amine amine groups groups present present in in the the dendrimer dendrimer with with copper copper sulfate, sulfate, in in half half generation, generation, reaction the blue color of copper sulfate was observed signifying the absence of terminal amine groups. The the blue color of copper sulfate was observed signifying the absence of terminal amine groups. Ultraviolet-visible (UV-Vis) spectrometry further provided the proof of synthesis by showing The Ultraviolet-visible (UV-Vis) spectrometry further provided the proof of synthesis by showing changes in in the the wavelength wavelength values values of of the the dendrimers. dendrimers. The wavelength of of the the synthesized synthesized changes The absorption absorption wavelength dendrimer changed from λ max 310 to 299, 291, 287, 279, 264, and 250 nm for G1, G1.5, G2, G2.5, G3, G3, dendrimer changed from λmax 310 to 299, 291, 287, 279, 264, and 250 nm for G1, G1.5, G2, G2.5, G3.5 and and G4, G4, respectively. respectively. The The change change in in wavelength wavelength was was due due to to the the changes changes in in the the structure structure of of the the G3.5 dendrimer generations. While the PAMAM dendrimer generation increased, the absorption shifted dendrimer generations. While the PAMAM dendrimer generation increased, the absorption shifted from high high to to lower lower wave wave numbers. numbers. from The Infrared Infrared (IR) (IR) spectrum spectrum of of aa solid solid EDTA, EDTA, aa synthetic synthetic starting starting material material was was acquired acquired parallel parallel The with that of the synthesized PAMAM G4 dendrimer for comparison purpose (Figure 2). The IR IR with that of the synthesized PAMAM G4 dendrimer for comparison purpose (Figure 2). The −1 ´ spectrum of the EDTA showed absorption due to the presence of carboxylic carbon at 1680.9 cm spectrum of the EDTA showed absorption due to the presence of carboxylic carbon at 1680.9 cm 1 .. −1 The IR spectrum of the synthesized dendrimer G4 showed absorption peaks at 3353.4 cm for N–H stretching of primary amine, 3277.4 and 3182.4 cm−1 for N–H stretching of secondary amine. The peaks at 2927.9 cm−1 and 2856.5 cm−1 were for26364 aliphatic C–H stretches. A peak at 1654.5 cm−1 was 2

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The IR spectrum of the synthesized dendrimer G4 showed absorption peaks at 3353.4 cm´1 for N–H stretching of primary amine, 3277.4 and 3182.4 cm´1 for N–H stretching of secondary amine. The peaks at 2927.9 cm´1 and 2856.5 cm´1 were for aliphatic C–H stretches. A peak at 1654.5 cm´1 Int. J. Mol. Sci. 16, page–page was assigned for2015, amide carbonyl absorption while a peak at 1568.4 cm´1 was due to a core N–C stretching. A disappearance of the IR absorption band at 1680.9 cm´1 for carboxylic carbon for EDTA assigned for amide carbonyl absorption while a peak at 1568.4 cm−1 was due to a core N–C Int. J. Mol. Sci. 2015, 16, page–page and appearance of IR absorption at 1654.5 cm´1 for the PAMAM G4 dendrimer was a further evidence stretching. A disappearance of the IR absorption band at 1680.9 cm−1 for carboxylic carbon for EDTA of the assigned success of the synthetic transformation carried out. −1 was due to a core N–C for amide carbonyl absorption while peak 1568.4 cm and appearance of IR absorption at 1654.5 cm−1a for theatPAMAM G4 dendrimer was a further −1 The Matrix-Assisted Time Mass Spectrum (MALDI-TOF stretching. disappearance the IR absorption band atcarried 1680.9of cmFlight for carboxylic carbon for EDTA evidence ofAthe successLaser of theofDesorption/Ionization synthetic transformation out. and appearance absorption at 1654.5 cm−1 for the PAMAM G4 dendrimer further ion MS) shown inMatrix-Assisted Figureof3,IR does not show appreciable peaks at region expected for amolecular The Laser Desorption/Ionization Time of the Flight Mass Spectrumwas (MALDI-TOF evidence oftothe success3,ofdoes the synthetic carried out. MS) shown intheoretical Figure not Wt. showtransformation appreciable peaks at the expected for molecular corresponding Mol. (14.21 kDa), instead itregion contains a broad peak at ion a region The Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrum (MALDI-TOF corresponding to theoretical Mol. Wt. (14.21 kDa), instead it contains a broad peak at a region ideal ideal for half molecular mass for PAMAM G4 (approximately 7.10 kDa) corresponding to [M]2+ . MS)half shown in Figure 3, does not showG4 appreciable peaks7.10 at the region expected for ion molecular mass for PAMAM (approximately kDa) corresponding tomolecular [M]2+defects . Similar Similarforphenomenon including molecular mass deviations and dendrimer structural under corresponding to theoretical Mol. Wt. (14.21 kDa), instead it contains a broad defects peak at under a region ideal phenomenon including molecular mass deviations and dendrimer structural MALDI MALDI MS determination has been reported [15,16]. Missing molecular ion and peak broadening 2+. Similar for molecular mass for reported PAMAM[15,16]. G4 (approximately 7.10 kDa) corresponding to [M]at MShalf determination has been Missing molecular ion and peak broadening half-size at half-size for the desired molecular mass is ascertained to random fragmentations applied phenomenon including molecular deviations dendrimer structuralunder defectsapplied underunder MALDI for the desired molecular mass ismass ascertained to and random fragmentations MALDI 2+ indicates possible presence of MALDI operating energy, the latter observation coinciding with [M] 2+ MS determination reported [15,16].coinciding Missing molecular ion and peak broadening at half-size operating energy,has thebeen latter observation with [M] indicates possible presence of for the desired molecular mass is ascertained to random fragmentations under applied MALDI as-synthesized PAMAM G4.G4. as-synthesized PAMAM operating energy, the latter observation coinciding with [M]2+ indicates possible presence of 1.1 as-synthesized PAMAM G4.

Transmittance (%) Transmittance (%)

1.1 1

1 0.9

0.9 0.8

0.8 0.7 PAMAM-G4 EDTA core

0.7 0.6 4000

3500

PAMAM-G4 3000 core2500 EDTA

2000

1500

1000

500

Wavenumber (cm ) 2500 2000 1500

1000

500

-1

0.6 4000

3500

3000

Figure 2. The superimposed IR spectra of solid EDTA (green) and PAMAMG4 (blue).

Intens. [a.u.]Intens. [a.u.]

-1 (cm ) Figure 2. The superimposed IR spectraWavenumber of solid EDTA (green) and PAMAMG4 (blue).

Figure 2. The superimposed IR spectra of solid EDTA (green) and PAMAMG4 (blue).

[M]2+ 300

[M]2+

300

200

200

100

100

0 2500

5000

7500

10000

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15000

17500

20000

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m/z 0

Figure 25003. MALDI-TOF of 15000 PAMAM 17500 G4 acquired 22500 using 5000 7500 mass 10000spectrum 12500 20000 m/z Trans-2-(3-(4-tert-butylphenyl)-2-methyl-2-propenylidene)malononitrile (DCTB) as a matrix. Figure 3. MALDI-TOF mass spectrum of PAMAM G4 acquired using FigureTrans-2-(3-(4-tert-butylphenyl)-2-methyl-2-propenylidene)malononitrile 3. proton MALDI-TOF mass resonance spectrum (1of PAMAM G4 of acquired Trans-2-(3-(4-tertThe nuclear magnetic H NMR) spectrum PAMAM dendrimer (Figure (DCTB)using asG4 a matrix.

butylphenyl)-2-methyl-2-propenylidene)malononitrile (DCTB) as a matrix. 4) consisted of set of signals due to only six different protons correlated to partial structure in Figure The proton nuclear magnetic resonance (1H NMR) spectrum of PAMAM G4 dendrimer (Figure 3 protons correlated to partial structure in Figure 4) consisted of set of signals due to only six different

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resonance (1 H NMR) spectrum of PAMAM G4 dendrimer (Figure 4) consisted of set of signals due to only six different protons correlated to partial structure in 5. The proton peaks were designated as H andand Hf.HThe most deshielded peak Figure 5. The proton peaks were designated asa,HHab, ,HHbc,, HHcd, ,HHde, H e f . The most deshielded peak resonating at δ 4.02 was assigned to amide protons (H a) while the proton signal resonating at δ 3.48 resonating at δ 4.02 was assigned to amide protons (Ha ) while the proton signal resonating at δ 3.48 was assigned to terminal amine protons (Hb). The proton peak resonating at δ 1.93 was assigned to was assigned to terminal amine protons (Hb ). The proton peak resonating at δ 1.93 was assigned methylene protons near the carbonyl group (Hc). Furthermore, the peak at δ 1.72 was assigned to to methylene protons near the carbonyl group (Hc ). Furthermore, the peak at δ 1.72 was assigned to methylene protons next to the amido nitrogen (Hd). An upfield peak resonating at δ 1.69 was methylene protons next to the amido nitrogen (Hd ). An upfield peak resonating at δ 1.69 was assigned assigned to methylene protons (He) adjacent to the terminal amine. The most shielded upfield peak to methylene protons (He ) adjacent to the terminal amine. The most shielded upfield peak resonating resonating at δ 1.35 was assigned to methylene protons inside the core (Hf) and those near tertiary at δamine 1.35 was (Hf′).assigned to methylene protons inside the core (Hf ) and those near tertiary amine (Hf1 ).

7.5

7.0

6.5

6.0

5.5

5.0

4.5

4.0 3.5 f1 (ppm)

0.07

e

f f´

2.4 3.4 2.7 3.9

3.50 3.50 3.4 9 3.48 3.47 3 .46

1.94 1.92 1.91 1.71 1.68 1.68

c

b

1.4

1.0

4 .03 4.01

a

d

1.37 1.33 1.13 1.11 1.10

7.26 cdcl3

1.56 HDO

Int.The J. Mol.proton Sci. 2015, 16, page–page nuclear magnetic

3.0

2.5

2.0

1.5

1.0

0.5

0.0

Figure 4. The 1H NMR spectrum of PAMAM dendrimer G4 observed at 400 MHz for CDCl3 solution Figure 4. The 1 H NMR spectrum of PAMAM dendrimer G4 observed at 400 MHz for CDCl3 solution at 25 °C. at 25 ˝ C.

The structural analysis of synthesized dendrimer G4 as represented by atom labeled partial 1H NMR spectrum The structural of synthesized G4 as represented by structure atom labeled partial structure (Figure 5)analysis is in agreement with thedendrimer (Figure 4). The consists of structure (Figure 5) is agreement the 1 Hprotons NMR spectrum (Figure The structure consists six different types ofin protons. Thesewith are amido (a), protons at the4). terminal amino group (b), of sixmethylene different types of protons. are amido protons (a), protons at the terminal amino group protons adjacent These to carbonyl (c), methylene protons adjacent the amido nitrogen (d),(b), methyleneprotons protonsadjacent next to terminal amino(c), group (e), methylene nearthe theamido tertiarynitrogen amine (fʹ)(d), methylene to carbonyl methylene protonsprotons adjacent and the methylene protons of the ethylenediaminetetraacetic acid core (f). Generally, results from(f1 ) methylene protons next to terminal amino group (e), methylene protons near the tertiary amine thethe UV-Vis, IR, NMR and MALDI-TOF-MS corroborated those acid reported previous studies [15–20]. and methylene protons of the ethylenediaminetetraacetic corein(f). Generally, results from Therefore, with these evidences, EDTA core PAMAM G4 dendrimer was successfully synthesized the UV-Vis, IR, NMR and MALDI-TOF-MS corroborated those reported in previous studies [15–20]. and characterized. Therefore, with these evidences, EDTA core PAMAM G4 dendrimer was successfully synthesized and characterized.

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b NH2b NH2 d d ee O O NH a NH a e c O a e c f O d e a NH N f N N N e NH d N NHd N e aNH f' N f N c d c f e O f' a 2 O 2 NH O NH O NH2 NH2

core core

braching unit braching unit

terminal group terminal group

Figure 5. Chemical structure and atom labeling of the part of dendrimer G4. Figure G4. Figure5.5.Chemical Chemicalstructure structureand andatom atomlabeling labelingofofthe thepart partofofdendrimer dendrimer G4.

2.2. Encapsulation and Characterization of Tetramethylscutellarein (TMScu (1))-Dendrimer Complex 2.2. 2.2.Encapsulation Encapsulationand andCharacterization CharacterizationofofTetramethylscutellarein Tetramethylscutellarein(TMScu (TMScu(1))-Dendrimer (1))-DendrimerComplex Complex The encapsulation ability of amine terminated PAMAM G4 dendrimer by complexing with The Theencapsulation encapsulationability abilityofofamine amineterminated terminatedPAMAM PAMAMG4 G4dendrimer dendrimerbybycomplexing complexingwith with tetramethylscutellarein (TMScu, 1) was investigated using IR, NMR spectroscopic and in vitro release tetramethylscutellarein (TMScu, 1) was investigated using IR, NMR spectroscopic and in vitro release tetramethylscutellarein (TMScu, 1) was investigated using IR, NMR spectroscopic and in vitro release studies. The superimposed IR spectra of TMScu (1) and the compound–dendrimer complex are shown studies. superimposedIR IRspectra spectra TMScu the compound–dendrimer complex are studies. The superimposed of of TMScu (1) (1) andand the compound–dendrimer complex are shown in Figure 6. Compound 1 exhibited IR absorption peak at 1626.4 cm−1 −1corresponding to aromatic ´1 corresponding shown in Figure 6. Compound 1 exhibited IR absorption peak at 1626.4 cm to in Figure 6. Compound 1 exhibited IR absorption peak at 1626.4 cm corresponding to aromatic to aromatic ring C–H conjugated carbonyl group. The absorption peak at 3075.1 cm−1 −1was assigned ´ 1 aromatic conjugated group. The absorption 3075.1 cmassigned was assigned to aromatic was to aromatic ring C–H conjugated carbonylcarbonyl group. The absorption peak at peak 3075.1atcm stretch. In PAMAM G4-1 complex, a new broad absorption peak appeared between 3500–3100 cm−1,−1 ring C–HInstretch. PAMAM G4-1 complex, new broadpeak absorption appeared between stretch. PAMAMInG4-1 complex, a new broadaabsorption appearedpeak between 3500–3100 cm , which indicated interaction between the dendrimer and the encapsulated compound. The ´1 , the 3500–3100 cm which indicated the interaction between the dendrimer and the encapsulated which indicated the interaction between the dendrimer and the encapsulated compound. The in the region around 3324.1 formation and existence of a broad peak (3500–3100 cm−1)−1with a loop ´ compound. The formation and existence of (3500–3100 a broad peak cm 1in ) with a loop around in the region ) with a loop the region 3324.1 formation and existence of a broad peak cm(3500–3100 −1 cm −1in the IR spectra of PAMAM G4-1 complex suggests the presence of –NH3+ functionality which+ ´1 in around IR spectra PAMAM G4-1 complex suggests the 3+presence of –NH cm in3324.1 the IR cm spectra ofthe PAMAM G4-1ofcomplex suggests the presence of –NH functionality which 3 results from electrostatic interaction between compound 1 and the dendrimer. Such broadening in functionality which results from electrostatic interaction between compound 1 and the dendrimer. results from electrostatic interaction between compound 1 and the dendrimer. Such broadening in comparable to previously reported IR in the region of NH and NH2 due to formation of –NH3+ is + is comparable + is comparable Such in and the region of NH and NH2ofdue to 3formation of –NH to to formation –NH to3 previously reported IR inbroadening the region in of IR NH NH2 due encapsulation studies [5,21]. previously reported encapsulation studies [5,21]. encapsulation studies [5,21]. 1.2 1.2

Transmittance (%) Transmittance (%)

1

1

0.8 0.8 0.6 0.6

0.4 0.4 0.2 0.2 4000 4000

TMScu (1) TMScu (1) PAMAM-G4-46complex PAMAM-G4-46complex

3500 3500

3000 2500 2000 1500 -1 3000 Wavenumver 2500 2000 1500 (cm ) -1 Wavenumver (cm )

1000 1000

500 500

Figure 6. The superimposed IR spectra of TMScu (1) and its dendrimer complex. Figure Figure6.6.The Thesuperimposed superimposedIRIRspectra spectraofofTMScu TMScu(1)(1)and anditsitsdendrimer dendrimercomplex. complex.

The 1H1 NMR spectra of pure compound 1 and its dendrimer complex are presented in Figures 7 The1 H H NMR ofofpure compound 1 and its dendrimer complex are presented in Figures The NMR spectra spectra pure compound its spectrum dendrimer are (PAMAM presented in 7 NMR of complex the complex G4 and 8, respectively. As indicated in Figure 7, the 11H1and 1 H NMR spectrum of the complex (PAMAM G4 and 8, respectively. As indicated in Figure 7, the Figures (1)) 7 and 8 respectively. indicated in Figure 7, the NMR spectrum1.ofThe the complex (PAMAM TMScu contains signals As from both the PAMAM G4 H and compound appearance of the TMScu (1)) contains signals from both the PAMAM G4 and compound 1. The appearance of the G4 TMScu contains signals from both1the G4 and compoundcomplex 1. The appearance of the slight shift (1)) of some peaks of compound in PAMAM the compound–dendrimer as illustrated by slight shift of some peaks of compound 1 in the compound–dendrimer complex as illustrated by stacked 1H1 NMR spectra (Figure 9a,b) suggests interactions brought about by an external electrostatic stacked H NMR spectra (Figure 9a,b) suggests interactions brought about by an external electrostatic interaction between TMScu (1) and PAMAM. The methoxy protons showed significant signal 26367 interaction between TMScu (1) and PAMAM. The methoxy protons showed significant signal 5 5

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slight shift of some peaks of compound 1 in the compound–dendrimer complex as illustrated by stacked 1 H NMR spectra (Figure 9a,b) suggests interactions brought about by an external electrostatic Int. J. Mol. Sci. 2015, 16, page–page interaction between TMScu (1) and PAMAM. The methoxy protons showed significant signal Int. J. Mol. Sci. 2015, 16, page–page dynamics with approximately δ 0.01 shifts and visually evident drifted signals showing doubling in dynamics with approximately δ 0.01 shifts and visually evident drifted signals showing doubling in PAMAM G4–TMScu complex (green, Figure 9b)visually suggesting interaction probably involving hydrogen dynamics approximately δ 0.01 shifts and drifted signals showing doubling in PAMAMwith G4–TMScu complex (green, Figure 9b) evident suggesting interaction probably involving bonding between methoxy oxygen(green, of 1 andFigure amino 9b) groups of the dendrimer G4. probably involving PAMAM G4–TMScu complex suggesting interaction hydrogen bonding between methoxy oxygen of 1 and amino groups of the dendrimer G4. hydrogen bonding between methoxy oxygen of 1 and amino groups of the dendrimer G4.

a OCH a 3 OCH3

b c

b

c

a H CO a3 H3CO

e

O

e

b

O

c

b

c 1.54 HDO 1.54 HDO

d

a aH3CO H3CO

1.5 4 1.5 4

Figure 7. The1 1H NMR spectrum of TMScu (1) observed at 499.88 MHz for CDCl3 solution at 25˝ °C. Figure H NMR NMR spectrum spectrum of of TMScu TMScu (1) (1) observed observed at at 499.88 499.88 MHz MHz for C. Figure 7. 7. The The 1H for CDCl3 CDCl3 solution solution at at 25 25 °C.

d OCH3 O a OCH3 O 1 a

1

a

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0 .07 0 .07

1.3 7 1 .341.3 7 1.141 .34 1 .111.14 1 .081 .11 1 .08

7.02 7.007.02 6.807.00 6.80 6.5 9 6.5 9

7.84 7.827.84 7.82 8.0

1.95 1.9 21.95 1.7 21.9 2 1.701.7 2 1.70 1 .6 8 1 .6 8

b d e b d e

c c

8.0

3.99 3.99 3.9 9 3.9 9 3 .98 33.98 .98 3.98 3.92 3.92 3.92 3.92 3 .89 33.89 .89 3.89 3.4 8 3.4 8

a

0.5 0.5

0.0 0.0

Figure 8. The 1H NMR of spectrum of PAMAM G4-TMScu (1) complex observed at 400 MHz for 1 Figure 8.solution The for CDCl38. 25 °C.of Figure The 1HHatNMR NMR of spectrum spectrum of of PAMAM PAMAM G4-TMScu G4-TMScu (1) (1) complex complex observed observed at at 400 400 MHz MHz for CDCl 3 solution at 25 °C. ˝ CDCl solution at 25 C. 3

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(a)

2 G 4 E S L M 3 _ P R O T O N _ 0 0 1 _ c d c l3

2 G 4 E S L M 3 _ P R O T O N _ 0 0 1 _ c d c l3

2

x

x

x

x

J M _ 4 E S L M 3 _ P R O T O N _ 0 0 1 _ c d c l3

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3.95

3 .9 4 f1 ( p p m )

3.93

3 .9 2

3 .9 1

3 .9 0

3.8 9

3 .8 8

3 .8 7

(b) Figure 9. (a) An expansion of the 1H NMR of spectrum of TMScu (1) encapsulated in PAMAM G4

Figure 9. (a) An expansion of the 1 H NMR of spectrum of TMScu (1) encapsulated in PAMAM G4 complex (green) stacked with the 1H NMR of spectrum of TMScu (1) without PAMAM G4 dendrimer 1 H NMR of spectrum of TMScu (1) without PAMAM G4 dendrimer complex (green) stacked with the observed at 400 MHz for CDCl3 solution at 25 °C; (b) An expansion of the 1H NMR of spectrum of TMScu 1 H NMR of spectrum observed at 400 MHz for CDCl3 at 25 ˝stacked C; (b) An ofofthe (1) encapsulated in PAMAM G4solution complex (green) withexpansion the 1H NMR spectrum of TMScu (1) 1 H NMR of spectrum of TMScu (1)PAMAM encapsulated in PAMAM G4 atcomplex stacked with the without G4 dendrimer observed 400 MHz(green) for CDCl3 solution at 25 °C (x = drifted signals of TMScu (1) without PAMAM G4 dendrimer observed at 400 MHz for CDCl3 solution at 25 ˝ C illustration). (x = drifted signals illustration). 2.3. Encapsulation Efficiency (EE) and Loading Capacity (LC)

2.3. Encapsulation Efficiency (EE) and Capacity (LC) Encapsulation efficiency (EE)Loading of compound 1 (C 19H18O6, M = 342.3 g/mol) was 77.8% ± 0.69%, while its loading capacity (LC) was 6.2% ± 0.06%. The encapsulation efficiency is influenced by

Encapsulation efficiency (EE) of compound 1 (C19 H18 O6 , M = 342.3 g/mol) was 77.8% ˘ 0.69%, among other factors the nature of the polymer nanoparticle and the encapsulated drug molecules, whilepolymer–drug its loading capacity was 6.2% ˘ [1,22]. 0.06%.TheThe encapsulation is influenced ratio and(LC) the concentration value obtained for efficiency encapsulation efficiency by among other factors the could nature the polymer nanoparticle and the encapsulated drug molecules, and loading capacity beof attributed to the intrinsic features of both PAMAM G4 dendrimer and polymer–drug and intermolecular the concentration [1,22]. Furthermore, The value obtained for encapsulation efficiency TMScu (1) ratio enabling interactions. dendrimers with high molecular weight such as PAMAM G4attributed would have loading capacity encapsulation as and loading capacity could be to high the intrinsic features and of both PAMAM capacity G4 dendrimer opposed to those with lower molecular weight [22]. The phenomenon is also explained by the results and TMScu (1) enabling intermolecular interactions. Furthermore, dendrimers with high molecular of such the present study, G4 the would EDTA core dendrimer high molecular weight as well as larger core weight as PAMAM haveG4 high loadinghas capacity and encapsulation capacity as opposed size and thus has higher loading capacity as observed. to those with lower molecular weight [22]. The phenomenon is also explained by the results of the present study, the EDTA core G4 dendrimer has high molecular weight as well as larger core size and thus has higher loading capacity as observed. 2.4. Phase Solubility Studies of the TMScu (1)-Dendrimer G4 Formulation 7

Measurements of solubilization ability of PAMAM dendrimer TMScu (1) was done, as previously described [5]. Phase solubility diagram was obtained by plotting the equilibrium 26369

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Measurements of solubilization ability of PAMAM dendrimer TMScu (1) was done, as previously described [5]. Phase solubility diagram was obtained by plotting the equilibrium concentration of compound 1 against PAMAM dendrimer concentrations (Figure 10). The solubility concentration of compound 1 against PAMAM dendrimer concentrations (Figure 10). The solubility increased linearly as function of PAMAM dendrimer concentration albeit with some deviations increased linearly as function of PAMAM dendrimer concentration albeit with some deviations noticeable at higher concentrations of the dendrimer at pH 7.2. According to the model developed noticeable at higher concentrations of the dendrimer at pH 7.2. According to the model developed by by Higuchi and Connors [23], the linear relationship between TMScu (1) solubility and PAMAM Higuchi and Connors [23], the linear relationship between TMScu (1) solubility and PAMAM dendrimer concentration indicated an A -type phase diagram particularly at pH 4. The obtained dendrimer concentration indicated an ALL-type phase diagram particularly at pH 4. The obtained linear diagrams (Figure 9) suggest the formation of water-soluble complex in solution due to linear diagrams (Figure 9) suggest the formation of water-soluble complex in solution due to electrostatic and hydrogen bonding interactions. Such an A -type phase solubility profile is electrostatic and hydrogen bonding interactions. Such an AL-type Lphase solubility profile is obtained obtained when the complex is first order with respect to both the dendrimer (ligands) and the when the complex is first order with respect to both the dendrimer (ligands) and the drug (substrate) drug (substrate) [23,24]. Generally, the solubility of drug–dendrimer complex increases linearly [23,24]. Generally, the solubility of drug–dendrimer complex increases linearly with increasing with increasing dendrimer concentration due to electrostatic interactions between the interactive dendrimer concentration due to electrostatic interactions between the interactive functional groups functional groups of the drug and the amino groups of the dendrimer [3,25]. The reported mechanism of the drug and the amino groups of the dendrimer [3,25]. The reported mechanism of interaction of interaction could hold the same for compound 1, as it contains the carbonyl and methoxy could hold the same for compound 1, as it contains the carbonyl and methoxy functionalities in its functionalities in its structure. Thus, the electrostatic interaction between the primary amine of structure. Thus, the electrostatic interaction between the primary amine of PAMAM G4 and the PAMAM G4 and the carboxyl or methoxy groups of the investigated compound could be responsible carboxyl or methoxy groups of the investigated compound could be responsible for its enhanced for its enhanced solubilization. Furthermore, at pH 4.0 can differences be attributed to athe solubilization. Furthermore, low solubility at pHlow 4.0 solubility can be attributed to the in pK of differences in pK of PAMAM dendrimer at different pH levels. The study by Jun-Jun et al., found a PAMAM dendrimer at different pH levels. The study by Jun-Jun et al., found that the amino groups that the amino groups the dendrimer pKPAMAM surface of amines PAMAM dendrimer a [25]. Thedendrimer of the dendrimer have of different pKa [25].have Thedifferent surface of has pKa that amines has pK that ranges from 7 to 9 while that of tertiary amines ranges from 3 to 6 [25]. Hence a ranges from 7 to 9 while that of tertiary amines ranges from 3 to 6 [25]. Hence as for the present study, as for the present the difference in pKa of the PAMAM G4 dendrimer would suggest that the difference in pKstudy, a of the PAMAM G4 dendrimer would suggest that dissociation would change dissociation would change with variation of pH leading towith the variation of solubility with pH. with variation of pH leading to the variation of solubility pH.

Concetration of TMScu (1) (M)

1 0.9 0.8 0.7 0.6 0.5

At pH 4.0

0.4

At pH 7.2

0.3 0.2 0.1 0 0

1 2 3 Concetration of PAMAM (M)

4

5

Figure 10. Phase solubility diagram of TMScu (1) in the presence of increasing PAMAM Figure 10. Phase solubility diagram of TMScu (1) in the presence of increasing PAMAM concentration. concentration.

2.5. In In Vitro Vitro Release Release of of the the Loaded Loaded TMScu TMScu (1) (1) 2.5. In vitro vitro release release to to investigate investigate the the stability stability and and interaction interaction of of TMScu-dendrimer TMScu-dendrimer complex complex was was In done in in aa phosphate phosphate buffered buffered saline saline (PBS) (PBS) media media at at two two different different pH In vitro vitro done pH values values (4.0 (4.0 and and 7.0). 7.0). In release behavior of loaded compound is presented in Figure 11. For a period of 1 h at pH 4.0, release behavior of loaded compound is presented in Figure 11. For a period of 1 h at pH 4.0, TMScu TMScu (1) showed an initial release of 17%, which later increased up to 36% for a period of 4 h. (1) showed an initial release of 17%, which later increased up to 36% for a period of 4 h. The release The nearly releasemaintained was nearlyfor maintained forto3 the h (i.e., up to the and seventh and thenincreased. progressively was 3 h (i.e., up seventh hour) thenhour) progressively The increased. The highest release of 75% was obtained after 13 h. However, at pH 7.0, the (1) release highest release of 75% was obtained after 13 h. However, at pH 7.0, the release of TMScu was of TMScu (1) awas onlyof22% forAs a period of 13 h. As 11, indicated Figure 11, ofthethein investigated vitro release only 22% for period 13 h. indicated in Figure the in in vitro release of the investigated pH anddependent. ionic interaction dependent. This gives compound was pHcompound and ionic was interaction This gives an implication thatanatimplication pH 7 the that at pH 7 the TMScu–dendrimer complex formulation was relatively stable as TMScu–dendrimer complex formulation was relatively stable as few percentages offew thepercentages compound of thereleased compound were released asatcompared that at pH 4. It is generally acknowledged were as compared to that pH 4. It istogenerally acknowledged that encapsulation of that the encapsulation of the compound by amine in basic condition prevents release from the dendrimer [26]. compound by amine in basic condition prevents release from the dendrimer [26]. Once the Once the dendrimer enter the cancerous cells or tumor cells which has acidic environment (low pH) the amino groups protonate, repel and undergoes8conformational change which facilitate the release 26370

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dendrimer enter the cancerous cells or tumor cells which has acidic environment (low pH) the amino groups protonate, repel and undergoes conformational change which facilitate the release of the of the compounds [26]. More molecules of TMScu (1) were released at pH 4.0, implying it could also compounds [26]. More molecules of TMScu (1) were released at pH 4.0, implying it could also be be made available to the targeted tumor cells’ physiological environment. made available to the targeted tumor cells’ physiological environment.

Figure 11. Release profile studies of loaded TMScu (1) from dendrimer–compound complex at pH 4 Figure 11. Release profile studies of loaded TMScu (1) from dendrimer–compound complex at and 7. pH 4 and 7.

2.6. Stability Studies of Dendrimer G4-TMScu (1) Formulation 2.6. Stability Studies of Dendrimer G4-TMScu (1) Formulation The study of the physical stability of the formulations at different temperature conditions, that The study of the physical stability of the formulations at different temperature conditions, that is, 0, 27 and 40˝°C to assess the disintegration was carried out and the results presented in Table 1. is, 0, 27 and 40 C to assess the disintegration was carried out and the results presented in Table 1. For For a period of two months, a sign of color changes, turbidity and precipitate formation were a period of two months, a sign of color changes, turbidity and precipitate formation were observed observed for formulation stored in light at 40 °C. The formulations/complexes were found to be for formulation stored in light at 40 ˝ C. The formulations/complexes were found to be relatively relatively stable, even at the warmer temperature of 40 °C when stored under dark conditions, stable, even at the warmer temperature of 40 ˝ C when stored under dark conditions, indicating that indicating that light had an influence on their stability. The formation of precipitates observed may light had an influence on their stability. The formation of precipitates observed may be attributed to be attributed to the opening of dendritic structures at higher temperatures [5]. There were no the opening of dendritic structures at higher temperatures [5]. There were no noticeable precipitates, noticeable precipitates, turbidity and color changes for the formulations stored in dark˝ and in light at turbidity and color changes for the formulations stored in dark and in light at 27 C, suggesting 27 °C, suggesting that the formulations should be stored in the dark, at low temperature or in a cool that the formulations should be stored in the dark, at low temperature or in a cool place for future place for future applications, should it be found useful. applications, should it be found useful. Table 1. Stability of PAMAM G4–TMScu (1) Formulation. Table 1. Stability of PAMAM G4–TMScu (1) Formulation.

Temperature (°C) Parameter Temperature Light(˝ C) Dark Light 0 Parameter Formulation 27 40 0Dark27 0 27 40 0 PAMAM G4-TMScu (1) Color change − − +++ − − PAMAM G4-TMScu (1) Color change Turbidity ´ −´ + +++ + −´ + Turbidity ´ + + ´ Precipitation −+ + ++++ −´ − Precipitation ´ Formulation

40 + ´ ++ + ´ +++ 27

40 + ++ +++

(−) No changes were observed, (+) changes were observed with varied extent. (´) No changes were observed, (+) changes were observed with varied extent.

3. 3. Experimental ExperimentalSection Section 3.1. 3.1. Materials Materials and and Reagents Reagents All sources and and used All chemicals chemicals and and reagents reagents were were obtained obtained from from commercial commercial sources used without without further further purification, unless otherwise stated. Chemicals and reagents included ethylenediaminetetraacetic purification, unless otherwise stated. Chemicals and reagents included ethylenediaminetetraacetic acid (Rochelle chemicals, chemicals, Johannesburg, Johannesburg, South South Africa, Africa, (SA), (SA), purity: purity: 99.5%), acid (EDTA) (EDTA) (Rochelle 99.5%), ethylenediamine ethylenediamine (EDA) (M & B, London, UK, purity: 98.5%), acetonitrile (Carloerba reagents, Avenue (EDA) (M & B, London, UK, purity: 98.5%), acetonitrile (Carloerba reagents, Avenue de de Valdonne, Valdonne, 13124 Peypin, France, purity: 99.5%), ethyl acetate (Fisher chemicals, Loughborough, Leics, UK, 9 26371

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13124 Peypin, France, purity: 99.5%), ethyl acetate (Fisher chemicals, Loughborough, Leics, UK, purity: 99.98%), methanol (Fischer chemicals), dicyclohexylcarbodiimide (DCC) (BDH Ltd., London, UK). Trans-2-(3-(4-tert-butylphenyl)-2-methyl-2-propenylidene)malononitrile (DCTB, 99.0%; Sigma-Aldrich, Stockholm, Sweden) Tetramethylscutellarein (TMScu, 1) was obtained from the Natural Products Research Laboratory, Chemistry Department, University of Dar es Salaam. Solvents used were analytical reagent (AR) grade. 3.2. General Experimental Procedures Reactions were run at room temperature unless stated otherwise. Purification and separation techniques included filtration, evaporation, and centrifugation. Glassware were washed and dried in an oven. The 1 H NMR spectra were obtained in CDCl3 using Varian MR 400 and Varian VNMR-S 500 at the Department of Chemistry and Molecular Biology, University of Gothenburg, Sweden. The IR spectra were recorded on Platinum ATR alpha Bruker spectrophotometry and UV-Vis spectroscopy measurements using UV-1800PC Spectophotometer (Shenandoah instruments, Inc., Beijing, China) and were performed at the Chemistry Department, University of Dar es Salaam. The autoflex MALDI-TOF-MS analysis was performed on FlexControl Bruker Daltonics, at Polymer Technolgy Laboratory, Chalmers University of Technology, Gothenburg, Sweden. DCTB was used as the matrix for the MALDI-MS acquisition. 3.3. Synthesis of Polyamidoamine (PAMAM) Dendrimer Generation 4 Ethylenediaminetetraacetic acid, EDTA core based PAMAM dendrimers were synthesized by divergent approach with reagent excess using Michael addition and amidation reactions [2]. The Michael addition involved the formation of half dendrimer generations, while in the amidation, two reactions steps were involved: (i) coupling reaction involving activation of carboxylic group and (ii) amidation step, which produced full generation amine terminated G4 PAMAM dendrimer (Scheme 1). In a typical experiment, EDTA core (6 g, 0.0205 mol) in a 250 mL flask was reacted with ethylenediamine (48 mL), EDA (2) in the presence of methanol as solvent (100 mL) and dicyclohexylcarbodiimide (4 g) (DCC, 4) for 48 h in dark while stirring in a tightly stoppered flask. The product obtained G1, was reacted with methacrylic acid (3) (30 mL) in the presence of methanol (100 mL) to give G1.5, and the solvent was evaporated by rotary evaporator. Apart from being a coupling and dehydrating agent, DCC was also used to activate the carboxylic group of EDTA. The dicyclohexylurea (DCU, 5), formed as a byproduct of DCC, was filtered and solvents were evaporated by a rotary evaporator. The activation step reaction was then followed by the amidation reaction (Scheme 2). In this step, the nitrogen lone pair from EDA acted as a nucleophile by reacting with the positive carbonyl carbon from the carboxylic group. These procedures were repeated in several steps to produce the fourth generation dendrimer (PAMAM G4). Synthesized PAMAM G4 was further characterized by IR, UV-Vis, 1 H NMR spectroscopy and MALDI-TOF-MS. 3.4. PAMAM G4 Yellowish oil: UV (λ max) 250 nm; IR (cm´1 ); 3350.71 (N–H stretching of primary amine), 3289.27 and 3182.38 (N–H stretching of secondary amine), 2931.22 and 2861.77 (aliphatic and asymmetric C–H stretching), 1643.79 (stretching of amide carbonyl) and 1565.49 (C–N stretching in a core). 1 H NMR (CDCl3 ): 4.02 ppm (–CONH–, Ha ); 3.48 ppm (–CONHCH2 CH2 NH2 , Hb ); 1.93 ppm (–CH2 CONH, Hc ); 1.72 ppm (–CONHCH2 CH2 –, Hd ); 1.69 ppm (NCH2 CH2 CONH–, He ); 1.35 ppm (–NCH2 CH2 N–, Hf and –N(tertiary)–CH2 CH2 , Hf1 ). 3.5. The Autoflex Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrum (MALDI-TOF-MS) Analysis The THF solution of the PAMAM G4 sample and DCTB matrix (1:10 v/v) was mixed in an Eppendorf tube. Then, 0.5 µL of the resulting solution was deposited on two spots on the stainless 26372

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microtiter format MALDI target and dried at room temperature to produce a solid layer [15]. The Int. J. Mol.mass Sci. 2015, 16, page–page MALDI-TOF spectra of PAMAM G4 dendrimer was acquired by 500 laser shots by autoflex when the instrument was operating in the positive ion linear (flight path 1.22 m) mode applying an when the instrument was operating in the positive ion linear (flight path 1.22 m) mode applying an Int. J. Mol. Sci. 2015, 16, page–page acceleration voltage of 20.04 kV. acceleration voltage of 20.04 kV. when the instrument was operating in the positive ion linear (flight path 1.22 m) mode applying an acceleration voltage of 20.04 kV.

Scheme 1. A divergent synthetic route for PAMAM G4 dendrimer.

Scheme 1. A divergent synthetic route for PAMAM G4 dendrimer. Scheme 1. A divergent synthetic route for PAMAM G4 dendrimer.

Scheme 2. Activation of carboxylic group by Dicyclohexylcarbodiimide (DCC) and amidation step reaction mechanism. Scheme 2. Activation of carboxylic group by Dicyclohexylcarbodiimide (DCC) and amidation step

Scheme 2. Activation of carboxylic group by Dicyclohexylcarbodiimide (DCC) and amidation step reaction mechanism. 11 reaction mechanism. 11

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3.6. Selection of Model Compound for Encapsulation Study The selected model compound (TMScu, 1) with a molecular weight of 342.3 (g¨ mol´1 ) was a UV active (at 314 nm) and sparingly soluble in aqueous media. 3.7. Encapsulation of TMScu (1) in PAMAM Dendrimer G4 Encapsulation studies of TMScu, 1 were done according to the reported methods [5,12]. An equal amount of drug to dendrimer ratio in grams was used. At first, 2 mg of compound 1 was dissolved in 10 mL of methanol (i.e., conditions that can dissolve 1), then 2 mg of the dendrimer was added. The reaction was stirred for 30 h and then the solvent was evaporated under vacuum to remove the methanol completely. This was followed by addition of deionized water to the complex and stirring for another 30 h. Water was filtered and freeze dried to afford a yellow powder of drug–dendrimer complex. Analysis of TMScu (1)–PAMAM–G4 complex was done by IR and NMR spectroscopic techniques to determine if there were absorbance changes or chemical shifts of the functional groups of the compound and/or dendrimer in the complex. 3.8. Encapsulation Efficiency (EE) and Loading Capacity (LC) To determine the encapsulation efficiency (EE) of the encapsulated compound 1 in the dendrimer, 2 mg of TMScu (1)–PAMAM complex was mixed with 5 mL methanol. The solution was then filtered using membrane of 0.22 µm pore size to exclude encapsulated from non-encapsulated TMScu (1) and analyzed by UV-Vis spectrophotometer at absorbance of 314 nm for compound 1. The encapsulation efficiency was calculated based on the concentration of the encapsulated compound detected in the formulation over the initial concentration of the compound in methanol as per Equation (1) [1]. rWt s ˆ 100% (1) Encapsulation efficiency pEE%q “ rWi s where W t is the total amount of compound detected in PAMAM formulation and W i is the total quantity of compound added initially during preparation. The loading capacity (LC) was also calculated based on Equation (2). Loading capacity pLC%q “

Weight of TMScu in PAMAM-G4 ˆ 100% Weight of PAMAM-G4

(2)

3.9. Solubilization Study of TMScu (1) Phase solubility studies of loaded compound (TMScu, 1) were done according to the previously reported method [5]. The studies were performed in pH of 4.0 and 7.2 to investigate the effect of pH on the solubility of the encapsulated compound. The pH 4.0 was chosen to reflect that of cancerous cells, while pH 7.2 is that of blood at normal physiological conditions. In the experiment, an excess of compound 1 was added into glass vials containing different concentration of PAMAM dendrimers (v/v 0.1%–0.4%) in pH 7.2 while in another vial containing only water, excess amount of the compound was added and treated as a control. Both vials were shaken at room temperature for 36 h in a shaker (Digital Grant Bio Shaker HY-5A, POS 300, USA) and allowed to stand attain equilibrium. The undissolved compound was removed by filtration through a 0.22 µm filter paper membrane size and washed twice with distilled water then solubilized in methanol and analyzed by UV-Vis spectrophotometer at the compound’s absorbance wavelength. A similar procedure was repeated at pH 4.0 for triplicate experiments and the mean values were used to plot the graphs. 3.10. In Vitro Release Studies of Loaded Compound (TMScu, 1) In vitro release studies of the loaded compound (TMScu, 1) from PAMAM dendrimer–TMScu complex were measured in triplicate in a PBS media at pH 7.0 and pH 4.0. One milligram of

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dendrimer–compound complexes was suspended in 5 mL of buffer solution (pH = 7.0 and 4.0) in a screw-capped vial. The vials were then placed in a shaker maintained at 37 ˝ C and shaken horizontally at 240 rpm at a predetermined time interval. The vials were then removed from the shaker and centrifuged for 10 min. The supernatant collected from the vials were then subjected for UV-Vis analyses. The precipitated dendrimer were re-suspended in 5 mL of fresh buffer solution and then put back into the shaker for continuous in vitro release measurements as per previously described method [21]. 3.11. Stability Studies of TMScu (1) The principles of chemical kinetics were employed in the stability studies of the formulation [5]. A stability study was therefore carried out under light and dark conditions with different temperature settings. In the stability study, 1 mL of formulations was kept in dark (using amber-colored glass vials) and in light (colorless vials) at 0, 27 ˝ C (room temperature) and 40 ˝ C in a controlled oven for 2 months. Samples were periodically withdrawn and analyzed for any color changes, crystallization, precipitation and turbidity. 4. Conclusions This study investigated the encapsulation and solubilization capacity of the as-synthesized PAMAM dendrimer G4 on a flavonoid tetramethylscutellarein (TMScu, 1) as an approach to enhance the potency of the flavonoid molecules. The synthesis of EDTA core based PAMAM dendrimer of generation four (G4) was designed and successfully achieved via a divergent approach by reacting EDA and acrylic acid in repeated steps. The synthesized G4 dendrimer was characterized by UV-Vis, IR and NMR spectroscopy. The encapsulation of the studied flavonoid (TMScu, 1) into PAMAM G4 dendrimer, solubilization and in vitro release were explored using a combination of IR, UV-Vis and NMR spectroscopic techniques. It was established that amine-terminated dendrimer G4 formed a complex with the flavonoid due to hydrogen bonding and electrostatic interaction as observed in the IR and NMR signal absorption changes. The amine terminated PAMAM G4 enhanced the solubility of the studied compound as a function of dendrimer concentration. Furthermore, pH was established to have an influence on the solubility with solubility being higher at pH 4 than at pH 7. The in vitro release studies indicated that PAMAM G4-MScu complex was relatively more stable in neutral medium at pH 7 than at pH 4. Overall, it has been established that PAMAM G4 dendrimer is capable of encapsulating TMScu and hence enhancing its solubility. The dendrimer thus has potential for the bioactivity enhancement of the studied and other related compounds warranting further investigation under in vivo and or in vitro bioassay setups. Acknowledgments: Authors wish to thank Mate Erdelyi of the Department of Chemistry and Molecular Biology, University of Gothenburg, Sweden for availing access to 1H NMR and MALDI-TOF mass spectra acquisition through existing collaboration with Stephen S. Nyandoro. Daniel M. Shadrack thanks the St. John’s University of Tanzania, for MSc fellowship through the Higher Education Loan Board (HESLB) fellowship scheme. Authors thank the University of Dar es Salaam, Chemistry department for providing bench space and analytical equipment. Author Contributions: Egid B. Mubofu and Stephen S. Nyandoro conceived and designed the experiments. Daniel M. Shadrack performed the experiments. All authors contributed to the preparation of the manuscript. Conflicts of Interest: The authors declare no conflict of interest.

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